Anticancer agent doxorubicin toxicity to in vitro X. laevis tadpole organ heart and mature frog heart and liver mitochondria

Abstract

One of the cancer drugs discharged into hospital wastewater, doxorubicin (DOX), is suspected of toxic effects on aquatic organisms. Cardiac and mitochondrial toxicity of DOX was investigated using cultured Xenopus (X.) laevis tadpole heart and mature frog liver mitochondria as materials. While 10^-9 DOX was not found to suppress heart rate, 10^-7 M DOX caused short-term heartbeat suppression in a preliminary experiment. Compared to the heart rate of untreated organ hearts kept in heart-organ medium without hydrogen-peroxide oxidoreductase enzyme catalase (CAT) for 9 days, that of 10^-8 M DOX-treated hearts decreased over time. The heartbeat suppression was improved by adding CAT to the heart culture medium, suggesting that DOX induces reactive oxygen species (ROS) in cultured tadpole hearts. Mitochondrial swelling assay was conducted. DOX was found to suppress slight swelling of heart and liver mitochondria with adenosine triphosphate (ATP) treatment. DOX also suppressed Ca⁺⁺-induced swelling of heart and liver mitochondria with ATP treatment. These findings suggest that the side effects of DOX on X. laevis heart and liver mitochondria are likely similar to those of cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore (MPT) and also a ROS generator, leading to cardiac and mitochondrial dysfunction.

INTRODUCTION

Human pharmaceuticals and their degradation products released into the environment through wastewater from hospitals and the other sources have been detected at low-level concentrations that may potentially have toxic effects on aquatic organisms in aquatic environments in many countries (Mahnik et al., 2007; Patel et al., 2019). Few reports regarding such substances have been evaluated using aquatic organisms, however. Various studies of the toxicity of pharmaceuticals released into the aquatic environment have been conducted using cultured cells derived from humans, mammals, cultured mammalian cells or aquatic organisms. For example, human pharmaceuticals have been reported to have adverse effects on Glandirana (G.) rugosa tadpole and Chinese hamster (Cricetulus barabensis griseus) ovary cell lines (Kitamura et al., 2005; Goto et al., 2006). Previously we also reported that the anti-arrhythmic drug amiodarone bioaccumulates in treated Xenopus (X.) laevis tadpoles and has a suppressive effect on spontaneous metamorphosis caused by endocrine disruption (Sanoh et al., 2020). Human pharmaceuticals released into the environment have a possibility to disrupt endocrine function in aquatic organisms, and anticancer drugs are no exception (Mahnik et al., 2007; Patel et al., 2019). DOX, one of the anticancer drugs used in the present study, has known toxic side effects, such as superoxide production in H9C2 myocyte from Rattus (R.) nortvegicus cardiomyocyte and HEK293 cell line derived from human (Homo (H.) sapiens) embryonic kidney and left ventricle of rats (Green and Leeuwenburgh, 2002; Ichikawa et al., 2014), cytochrome c (Cyt. c) release followed by caspase-3 activation, resulting in apoptosis of in vivo left ventricle of rats (Childs et al., 2002; Ueno et al., 2006). Thus, DOX actions that have been revealed to date are DNA-synthesis-blockage and ROS generator leading to apoptosis of human embryonic kidney and left ventricle of rats (Green and Leeuwenburgh, 2002; Ichikawa et al., 2014; Ueno et al., 2006). However, the mechanism of DOX-induced functional disruption of mitochondria remains unclear. With regard to the mechanism of Ca⁺⁺-induced mitochondrial swelling, in the review of apoptosis described by Javadov et al. (2018), they said that mitochondria under normal Ca⁺⁺ concentration are stably regulated, while mitochondria at high concentrations of Ca⁺⁺ can induce cell death pathways through non-selective mitochondrial membrane transition pore (MPTP) opening by reactive oxygen species (ROS) production in the presence of phosphate. Further, in the review of Zoratti and Szabò (1995), MPTP is thought to be composed of adenine nucleotide translocator (ANT) and voltage-dependent anion channel (VDAC). A cysteine group with ANT is oxidized by over-generated oxidative stress. ANT-VDAC then forms MPTP resulting in pore opening with associated mitochondrial swelling (Halestrap, 2009; Hanada et al., 2003; Zoratti and Szabò, 1995). Ca⁺⁺ sensitivity in this ANT-VDAC formation is enhanced by excessive oxidative stress, phosphate and adenine nucleotide depletion (Varanyuwatana and Halestrap, 2012). In other words, the cysteine group on ANT affected by excessive oxidative stress production is thought to be oxidized, and then forms a thiol group accompanied by conformational change, leading to MPT pore opening (Halestrap, 2009; Hanada et al., 2003; Zoratti and Szabò, 1995). The MPT pore opening formation in experiments using G. rugosa and rat liver is thought to be formed in CsA-sensitive apoptotic pathway, and called “classical” (Hanada et al., 2003; Mironova and Pavlov, 2021). On the other hand, Ca⁺⁺-stimulated energy-dependent K⁺ has induced matrix volume increase in rat liver mitochondria (Halestrap et al., 1986; Mironova et al., 2021). The swelling of mitochondria from rats and G. rugosa liver is also a dysfunctional condition caused by mitochondrial uncouplers and Ca⁺⁺ (Hanada et al., 2003). The swelling of mitochondria from rat liver is inhibited by ATP addition (Utsumi, 1963), and thereby mitochondria are able to maintain a more stable state. And the swelling of yeast mitochondria with ATP is induced by a medium containing the H⁺-ATP synthase inhibitor oligomycin, suggesting that ATP with oligomycin promotes the swelling of mitochondria from yeast (Saccharomyces cerevisiae) (Guérin et al., 1994). After mitochondrial swelling under uncoupler-stimulated conditions, Cyt. c leakage is thought to activate caspase proteases that can in turn induce apoptosis (Utsumi et al., 1965; Hanada et al., 1997; Kashiwagi et al., 1999; Arita et al., 2001; Kashiwagi et al., 2001). However, it is an important point that Ca⁺⁺ serves physiological functions in ATP synthesis, and mitochondrial swelling does not necessarily lead to cell and tissue death (Eisner et al., 2017; Nakano et al., 2011).

Cardiac toxicity, one of the side effects of DOX, has been confirmed (Childs et al., 2002). Severe damage to heart tissue by Cyt. c releasing from MPTP through superoxide production in chronically DOX-treated rats is suspected (Childs et al., 2002). Chronically DOX-treated rats display mitochondrial dysfunction in the heart, followed by ROS production and cardiac injury (Childs et al., 2002). Hanada et al. (2023) has reported that an in vitro X. laevis tadpole organ heart culture system has been developed. This culture system has two advantages: (1) the tadpole heart organ maintains long-term spontaneous pulsation, and (2) it allows for the testing of chemical substances (Hanada et al., 2023). In other words, this culture medium allows for observation of changes in tadpole-organ-heart rate that is directly influenced by DOX over a long term.

In the present study, DOX-induced heart dysfunction in X. laevis tadpole organ cultured heart, as well as side effects on X. laevis frog liver mitochondria in the presence of ATP and Ca⁺⁺ were confirmed.

MATERIALS AND METHODS

Chemicals

Doxorubicin hydrochloride was purchased from Tokyo Chemical Industry Co., Tokyo, Japan. Penicillin-streptomycin solution was purchased from Invitrogen Co. Ltd., San Diego, California, USA. Sulfathiazole, N-[2-hydroxyethyl]piperazine-N’-[2-ethanesulfonic acid] (HEPES), acetyl-L-carnitine and succinate were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Amphotericin B was purchased from HYCLONE^TM, USA. 50× MEM amino acids solution and 100× MEM vitamin solution were purchased from Gibco. Bovine serum albumin, CAT and glutamine were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Vitamin A acetate, Vitamin B₁₂, -C, -E, -D₃, -K₂, beef tallow, acetyl-L-carnitine, galactose, DNA from salmon testes and adenine were also purchased from Sigma-Aldrich, Inc. (St. Louis, Mo, USA). Sirius red was purchased from Muto Pure Chemicals Co. Ltd. 0.5% levobupivacaine hydrochloride was purchased from AstraZeneca PLC. Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (Mn(III)TMpyP) was purchased from Cayman chemical. Oleic acid was purchased from Tokyo Chemical Industry. Linoleic acid was purchased from Nacalai Tesque. Human chorionic gonadotropin (hCG) was purchased from ASKA Animal Health Co., Ltd. All reagents and frozen chicken hearts were purchased from Yamamoto Yakuhin Co., Ltd. and N.G.S. Co. Ltd.

Vitamin-A acetate, -E, -B₁₂, -E, -D₃, -K₂, linoleic acid and oleic acid dissolved in ethanol or DMSO were used. Adenine, glutamine and salmon DNA were dissolved in 0.5 M NaOH solution. Beef tallow was dissolved in an 83% dimethyl sulfoxide-17% ethanol solution, and a 10% solution was used as the stock solution.

Extraction of collagen from chicken heart, extraction of proteins from fetal bovine serum and blood pigment

Extraction of chicken heart collagens was according to a slightly modified methods of Liu et al. (2019) and Hanada et al. (2023) as follows. A frozen chicken heart cut into slices was immersed in 3% hydrogen peroxide (H₂O₂) for one day, and in turn, treated with 10 mM Tris-HCl buffered solution (pH 8.0) containing 0.8% Triton X-100 and 1 mM sodium ethylenediaminetetraacetate at room temperature for 2 more days. After treatment, one piece of chicken heart slice was picked up, and stained with picrosirius red stain solution to confirm the kind of collagen. After the slice was identified as collagen type I and III, with high-purity water and then washed at least 5 times. Collagens were dissolved in 2 or 4 M NaOH. Protein concentration of collagens was estimated using the Bradford method and stored at -20°C until use.

For the details of the extraction methods for fetal bovine serum and blood pigment, refer to the Materials and Methods section of Hanada et al. (2023).

Animals

Animals were treated according to the guideline “Regulations for animal experiments and related activities at Hiroshima University” which was established for the care and use of experimental animals by Hiroshima University (Permit number: G-22-6). All surgery was carried out under 0.5% levobupivacaine hydrochloride anesthesia, and thereby we made effort to reduce the suffering of the tadpoles and frogs.

Nieuwkoop and Faber (NF) (1956) stage 57 X. laevis tadpoles were used in the present study. Adult X. laevis frogs were derived from standard strains maintained by the Hiroshima University Amphibian Research Center. Mating was induced by injecting hCG into the dorsal lymph sac (males 60 U, females 250 U). Matings were carried out by separating individual male-female pairs into divided water tanks containing 4 L of chlorine-free tap water at 19-20°C for 10–20 hr. Tadpoles were maintained at 20-22°C, fed SERA Micron (Sera Heinsberg, Germany) and Tetramin (Spectrum Brand Japan & Spectrum Brand, Inc.), and staged according to Nieuwkoop and Faber.

Isolation of mitochondria from adult X. laevis heart and liver

Mitochondria were isolated from X. laevis frog heart and liver by the method of Hogeboom et al. (1948) and Kashiwagi et al. (2001) using sucrose density gradient centrifugation (Mustafa et al., 1966).

Briefly described, hearts and livers were cut out from the body and ground in a grinder on ice. 8 mL of 0.25 M sucrose solution (10 mM Tris-HCl buffer, pH 7.4) was poured into the mashed heart and liver, and suspended respectively. Suspensions were transferred into 6 or 8 centrifuge 14 mL tubes and centrifuged at 3200 rpm for 1 min in order to remove debris. Supernatants were recentrifuged at 10000 rpm for 10 min at 4°C. Transparent layers of the resultant liquids were then transferred to 1.5 mL tubes and stored at -20°C. Heart and liver mitochondria proteins were determined by the method of Bradford (1976) using bovine serum albumin as a standard. Mitochondria were measured at 540 nm absorbance using JASCO UVIDEC-320H.

Organ heart culture medium preparation for culturing and DOX testing

MHBSS-CM (Hanada et al., 2023) was modified by optional reduction of NaCl to induce a more stable anuran heart based on the composition of anuran leukocyte culture medium (Hanada, 2011, 2012). St. 57 tadpole hearts were cultured in a 1 mL solution composed of 2.8 g/L NaCl, 0.11 g/L CaCl₂, 0.026 g/L MgCl₂·6H₂O, 0.14 g/L KCl, 2×10^-4 M Na₂HPO₄, 5 mM HEPES, 44/1000-diluted 100× MEM vitamin solution, 2.5/1000000-diluted 50× MEM amino acids solution, 5 × 10^-7 M L-glutamine, 462 µg/dL collagen (average concentration), 0.02% sulfathiazole, 180 U/mL penicillin, 180 μg/mL streptomycin, 2.5 µg/mL amphotericin, 1.3% albumin, 850 μg/dL chicken blood pigment, 10^-5 M VA, 2.5×10^-5 M VE, 2.5×10^-5 M VC, 2×10^-3 mg/mL VB₁₂, 10^-4 mg/mL VD₃, 2×10^-3 mg/mL VK₂, 3.6×10^-4 M succinate, 0.8 mM acetyl-L-carnitine 391 µg/dL alkali solution of FBS protein, 10^-5 μL/mL linoleic acid, 0.5 µL/mL of beef tallow solution, 0.5 µL/mL of oleic acid and 2.5 µg/mL Mn(III)TMpyP.

The PH of all culture media was adjusted to 7.2 by adding 1 M NaOH or 1 M HCl. Subsequently culture media were sterilized by syringe with cellulose membrane filter (pore size 0.22 µm). After 1 hr, isolated hearts were further moved to new Petri dishes containing fresh culture medium to prevent contamination by various bacteria and mold. Culture media were replaced with fresh ones every tenth day. Organ-cultured hearts with morphological changes or no spontaneous heartbeat were excluded from the experiment. All tadpole hearts were used for pharmacological testing within 33 days after culture initiation.

Criteria for counting heartbeat

Observation of tadpole heart contraction/relaxation was conducted according to the method of Lajmanovich et al. (2019), Peltzer et al. (2019) and Hanada et al. (2023) with a slightly modified observation method. Observation of cultured hearts was conducted by binocular microscope (ZEISS stemi 305), and movies of the hearts were recorded by digital camera (Nikon digital sight 1000).

Sequential movements of sinus venosus, including pacemaker tissue, cardiac atriums, and ventricle, were monitored and counted as heartbeats on nontreated controls and DOX-stimulated hearts.

Assay for mitochondrial swelling

Experiments were conducted according to the slightly modified method of Hanada et al. (2003). Mitochondrial concentration in 1 mL of 10 mM Tris-HCl buffer pH 7.4 containing 0.15 M KCl was adjusted to 0.1 mg protein/mL. 0.2 mM dipotassium hydrogen phosphate and 1 mM succinate with mitochondria were added to 1 mL of Tris-HCl buffer pH7.4 containing 0.15 M KCl, and initiated with 1 mM ATP to stabilize mitochondrial membrane potential before chemical testing. Large amplitude swelling of mitochondria was monitored spectrophotometrically at 540 nm for 10 min using a spectrophotometer UVIDEC-320H (Japan Spectroscopic Co. Ltd.) in a thermostatically controlled room (22°C). A concentration of 5×10^-5 M DOX was confirmed to be effective in preliminary experiments.

Statistical analysis

Statistical analysis for Figs. 1, 2b, 3b, 4b and 5b was performed with R (R 3.6.3 for Mac OS ver. 10.15.7). Data for each figure was tested for homogeneity of variance using Levene’s test. The significance of differences between control and treated groups was evaluated by one-way analysis of variance (ANOVA) following a Dunnett’s post-hoc test. Results are expressed as mean +/- SEM. P-Values below 0.05 were considered statistically significant.

Fig. 1

Effect of DOX on cultured X. laevis organ hearts in the absence or presence of CAT. Control X. laevis organ hearts were not exposed to DOX or CAT throughout the experiment. Six X. laevis organ hearts were cultured in medium containing 10^-8 M DOX without CAT from initiation to day 9, at which point, and then 70 mU/mL CAT was added to each culture dish until the 26^th day the end. Open circle indicates controls (group 1), and closed circle indicates10^-8 M DOX-treated hearts (group 2). Values represent mean value +/- standard error. **Significantly less (P < 0.01) than corresponding control values. *Significantly less (P < 0.05) than corresponding control values.

Fig. 2

5×10^-5 M DOX-suppressed spontaneous swelling of mitochondria derived from the heart for 9 min after 1 mM ATP addition. (a) Effect of 5×10^-5 M DOX on mitochondrial swelling. DOX suppression of slight mitochondrial swelling for 9 min after 1 mM ATP addition. The solid line indicates control (group 1), and the dashed line indicates DOX treatment (group 2). Group 1: The control experiment was immediately initiated after 1, 0.2 mM potassium phosphate (Pi); 2, 1 mM succinate; 3, 0.1 mg protein/mL mitochondria; 4, 1 mM ATP addition were added in that order to the medium, and group 2: As mentioned above, DOX treatment: Mitochondria in the experiment were pretreated for 1 min after 5×10^-5 M DOX was added (a thin horizontal bar is seen in the figure), thereafter 1, 2, 3, 4, the reagents were added in that order to the medium, and then the experiment was immediately initiated after the addition of ATP to the medium. Addition timing of the reagents and mitochondria to the medium in control is similar to DOX treatment. Arrows shown in the figure indicate the timing of each added reagent and mitochondria. (b) Comparison values between group 1 (control) and group 2 (DOX-treated mitochondria) values. DOX significantly suppresses mitochondrial swelling in controls. Experiments were repeated 3 times. **Significantly greater (P < 0.01) than corresponding control values.

Fig. 3

5×10^-5 M DOX-suppressed spontaneous swelling of liver mitochondria for 8 min after 1 mM ATP addition. (a) Effect of DOX on mitochondrial swelling. 5×10^-5 M DOX-suppression of slight mitochondrial swelling for 8 min after 1 mM ATP addition. The solid line indicates control (group 1), and the dashed line indicates DOX treatment (group 2). Group 1: The control experiment was immediately initiated after 1, 0.2 mM potassium phosphate (Pi); 2, 1 mM succinate; 3, 0.1 mg protein/mL mitochondria; 4, 1 mM ATP addition were added in that order to the medium, and group 2: As mentioned above, DOX treatment: Mitochondria in the experiment were pretreated for 1 min after 5×10^-5 M DOX was added (a thin horizontal bar is seen in the figure), thereafter 1, 2, 3, 4, the reagents were added in that order to the medium, and then the experiment was immediately initiated after the addition of ATP to the medium. Addition timing of the reagents and mitochondria to the medium in control is similar to DOX treatment. Arrows shown in the figure indicate the timing of each added reagent and mitochondria. (b) Comparison values between group 1 (control) and group 2 (DOX-treated mitochondria) values. Box length indicates the interquartile range or IQR (25th to 75th percentile), and the horizontal line shows the median. The whiskers show distance 1.5 times IQR. Each experiment was repeated 6 times. *Significantly greater (P < 0.05) than the corresponding control values.

Fig. 4

5×10^-5 M DOX-suppressed Ca⁺⁺-induced swelling mitochondria from the heart for 6 min after 1 mM Ca⁺⁺ addition. (a) Effect of DOX on Ca⁺⁺-induced mitochondrial swelling. The solid line indicates group 3 (Ca⁺⁺), and the dashed line indicates group 4 (Ca⁺⁺ plus DOX). Group 3: control experiments were immediately initiated after 1, 0.2 mM Pi; 2, 1 mM succinate; 3, 0.1 mg protein/mL mitochondria; 4, 1 mM ATP, and then 1 mM Ca⁺⁺ were added to the medium. Group 4: As mentioned above, DOX treatment: Mitochondria in the experiment were pretreated for 1 min after 5×10^-5 M DOX was added (a thin horizontal bar is shown in the figure), thereafter 1, 2, 3, 4, the reagents were added in that order to the medium, and then the experiment was immediately initiated after the addition of Ca⁺⁺ to the medium. Arrows shown in the figure indicate the timing of each added reagent and mitochondria. (b) Comparison values between group 3 (Ca⁺⁺) and group 4 (Ca⁺⁺ plus DOX-treated mitochondria) values. DOX significantly suppresses mitochondrial swelling in controls. Each experiment was repeated 3 times. **Significantly greater (P < 0.01) than the corresponding positive control values.

Fig. 5

5×10^-5 M DOX-suppressed Ca⁺⁺-induced swelling mitochondria from the liver for 8.5 min after 1 mM Ca⁺⁺ addition. (a) Effect of DOX on Ca⁺⁺-induced mitochondrial swelling. The solid line indicates group 3 (Ca⁺⁺), and the dashed line indicates group 4 (Ca⁺⁺ plus DOX). Group 3: control experiments were immediately initiated after 1, 0.2 mM Pi; 2, 1 mM succinate; 3, 0.1 mg protein/mL mitochondria; 4, 1 mM ATP, and then 1 mM Ca⁺⁺ were added to the medium. Group 4: As mentioned above, DOX treatment: Mitochondria in the experiment were pretreated for 1 min after 5×10^-5 M DOX was added (shown by the thin horizontal bar in the figure), thereafter 1, 2, 3, 4, the reagents were added in that order to the medium, and then the experiment was immediately initiated after the addition of Ca⁺⁺ to the medium. Arrows shown in the figure indicate the timing of each added reagent and mitochondria. (b) DOX significantly suppresses mitochondrial swelling in controls. Each experiment was repeated 6 times. **Significantly greater (P < 0.01) than the corresponding positive control values.

RESULTS

DOX effect on organ-cultured X. laevis tadpole hearts

These experiments were carried out in order to clarify the suppressive effect of DOX on spontaneous heartbeat in cultured NF stage 57 X. laevis tadpole hearts. Fig. 1 shows the toxic effect of 10^-8 M DOX treatment on cultured hearts (n=6) for long-term heart rate. The heart rate for untreated controls (group 1) shows values between 1 and 1.2 relative to day 0, i.e., the stability of spontaneous heartbeats. The heartrate for DOX-treated hearts (group 2) showed a value of approximately 1 at 5 min after the initiation of DOX treatment, but then gradually decreased by nearly 0.4 relative to day 0 from day 5 to day 9. At this point 70 mU/mL CAT was added to DOX-treated hearts, and the heart rate gradually recovered. These results indicate that DOX induces dysfunction of the tadpole heart pacemaker, suggesting that DOX-induced ROS suppresses heart rate in cultured tadpoles.

Toxic effect of DOX on X. laevis frog mitochondria with inorganic phosphate and succinate treatment

Figs. 2a and b show the toxic effect of 5×10^-5 M DOX on heart mitochondria with inorganic phosphate and succinate treatment in a 0.15 M KCl-10 mM Tris-HCl buffer medium at pH 7.4. Fig. 2a shows that control heart mitochondria (group 1) were only slightly induced to swell over a period of 9 min, while DOX-treated heart mitochondria (group 2) displayed significantly suppressed mitochondrial swelling. Fig. 2b shows the comparison between the control group (group 1) and DOX-treatment group (group 2). DOX significantly suppressed mitochondrial swelling in controls. These results indicate the possibility that DOX has suppressive effect on swelling of frog heart mitochondria.

Contraction of slightly swollen mitochondria derived from the liver by DOX

Figs. 3a and b show the toxic effect of 5×10^-5 M DOX on liver mitochondria with inorganic phosphate and succinate treatment in a 0.15 M KCl-10 mM Tris-HCl buffer medium at pH 7.4. As shown in Fig. 3a, control liver mitochondria (group 1) were only slightly induced to swell over a period of 9.5 min, while DOX-treated liver mitochondria (group 2) displayed significantly suppressed swelling. Fig. 3b shows the comparison between the control group (group 1) and the DOX-treated group (group 2). DOX significantly suppresses mitochondrial swelling in controls. These results also indicate the possibility that DOX has a suppressive effect on swelling in frog liver mitochondria.

Suppressive effect of DOX on Ca⁺⁺-induced swelling of heart mitochondria

Figs. 4a and b show the toxic effect of 5×10^-5 M DOX on Ca⁺⁺-treated heart mitochondria with inorganic phosphate and succinate treatment in the medium of 0.15 M KCl-10 mM Tris-HCl buffer at pH 7.4. As shown in Fig. 4a, Ca⁺⁺ treatment alone induced mitochondrial swelling in controls (group 3). DOX presence in the medium tended to suppress mitochondrial swelling (group 4). Fig. 4b shows the comparison between Ca⁺⁺ (group 3) and Ca⁺⁺ plus DOX (group 4). These results indicate that DOX significantly suppresses Ca⁺⁺-induced swelling of heart mitochondria.

Suppressive effect of 5×10^-5 M DOX on Ca⁺⁺-induced swelling of liver mitochondria

Figs. 5a and b show the toxic effect of 5×10^-5 M DOX on Ca⁺⁺-treated liver mitochondria with inorganic phosphate and succinate treatment in a 0.15 M KCl-10 mM Tris-HCl buffer medium at pH 7.4. As shown in Fig. 5a, Ca⁺⁺ treatment alone induced mitochondrial swelling (group 3). DOX presence in the medium tended to suppress the mitochondrial swelling (group 4). Fig. 5b shows the comparison between Ca⁺⁺ only controls (group 3) and Ca⁺⁺ plus DOX (group 4). These results indicate that the presence of DOX in the medium significantly suppress Ca⁺⁺– induced swelling of liver mitochondria.

DISCUSSION

The present study found that one mechanism of in vitro functional X. laevis tadpole heart disorder involves DOX-induced mitochondrial dysfunction.

Symptoms of cardiotoxicity seen in chronically DOX-treated rats is marked by severe damage to heart tissue followed by Cyt. c releasing from mitochondrial membrane transition pore induced through superoxide production (Childs et al., 2002). Lee et al. (2023) reported that DOX suppressed glutathione activity, which in turn is thought to generate ROS.

Supplement Fig. 1 shows that 10^-7 M DOX treatment (group 2, n=3) resulted in a suppressed heart rate at 3.5 hr and 24 hr, compared to that of untreated controls (group 1). 10^-9 M DOX (group 2, n=3), on the other hand, had no significant effect on the heart rate in short term. These results indicate that a concentration-dependent increase in heart rate in short or long term. Short-term exposure of DOX to the cultured organ hearts was carried out in the present study, and the arrhythmia that occurred at 5 min after the initiation of DOX exposure lasted through day 9 when the experiment was stopped. In addition, the decreased heart rate seen in DOX-treated X. laevis tadpole hearts continued for an additional 17 days after the end of the experiment, at which point heart rate recovered with the addition of 70 mU/mL CAT to the medium, suggesting that DOX-induced hydrogen peroxide in the hearts was converted at 3.5 hr and 24 hr: 2H₂O₂ → 2H₂O + O₂. These results suggest that DOX-induced ROS production causes cardiac arrhythmia in cultured organ hearts.

Regarding the toxicity of DOX to mitochondria, it is well established that thyroid hormone induces anuran tadpole tail shortening through ROS production, and that tadpole tail shortening involves the mechanism of mitochondrial swelling and Cyt.c leakage leading to tail apoptosis, and that this is inhibited by CsA (Hanada et al., 1997; Kashiwagi et al., 1999; Hanada et al., 2003). CsA functions to inhibit mitochondrial swelling and Cyt. c leakage, and thus apoptosis. Ca⁺⁺ also induces swelling of rat liver and G. rugosa liver mitochondria, which is also inhibited by CsA (Hanada et al., 2003). Mitochondrial swelling caused by DOX-treatment in short term has not been addressed despite findings of Cyt.c leakage from the mitochondria of chronically DOX-treated rat left ventricle heart (Childs et al., 2002). In the present study, X. laevis liver mitochondria in the presence of ATP were slightly swollen, and this swelling was suppressed in the presence of DOX. More especially, the significant difference in swelling observed between DOX-treaed groups and controls indicates the unknown side effect of DOX toxicity shown in Figs. 2 and 3. In theory, mitochondrial swelling should be caused by DOX-induced ROS production, however, the side effect is able to suppress the swelling of X. laevis liver mitochondria, likely similar to CsA effect. In previous reports, isolated rat liver and guinea pig mitochondrial swelling is reported to be induced by Ca⁺⁺ (Strubbe-Rivera et al., 2021; Lee et al., 2023). He et al. (2020) reported that mitochondria isolated from cultured human umbilical vein endothelial cells are swollen with the addition of DOX in the presence of Ca⁺⁺. In the present study, Ca⁺⁺-induced X. laevis mitochondrial swelling was suppressed by DOX. This inhibition indicates the possibility that Ca⁺⁺ metabolism is disturbed in ANT on the inner mitochondria membrane of the organ-cultured heart. These results suggest that DOX induces the suppression of mitochondrial swelling, and resultant hydrogen peroxide production leads to heart arrhythmia in the in vitro tadpole organ heart.

In conclusion, DOX showed a cardiotoxicity that reduces pulsation of cultured organ hearts of X. laevis tadpoles through hydrogen peroxide production. Moreover, X. laevis frog heart and liver mitochondria with short-time treatment by DOX resulted in the suppression of slight or Ca⁺⁺-induced mitochondrial swelling. Mahnik et al. (2007) reported a DOX concentration range of 0.26 μg/L (0.48 nM) to 1.35 μg/L (2.48 nM) in wastewater monitored at a hospital with an oncology department. Concentrations in the aquatic environment are unknown, but given the possibility of bioaccumulation, there is concern about toxicological effects on tadpoles. Furthermore, it is also necessary to assess the mechanism, including ROS production, of possible DOX side effects in humans.

ACKNOWLEDGMENTS

X. tropicalis tadpoles were used to develop the culture medium, and were provided by Hiroshima University Amphibian Research Center, with support in part by the National Bio-Resource Project of the AMED, Japan. We are also grateful to Interuniversity Bio-Backup Project for Basic Biology for allowing us to use laboratory instruments. This research was partially supported by the Grants in‐Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grant number: 23K05490).

Conflict of interest

The authors declare that there is no conflict of interest.

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