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European Journal of Heart Failure 2009 11(2):154-162; doi:10.1093/eurjhf/hfn012
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2009. For permissions please email: journals.permissions@oxfordjournals.org.

Cyclophosphamide-evoked heart failure involves pronounced co-suppression of cytoplasmic thioredoxin reductase activity and non-protein free thiol level

Xufang Wang, Jinsong Zhang* and Tongwen Xu

School of Chemistry and Material Science, University of Science and Technology of China, Southern Campus, Meiling Avenue No.121, Hefei 230052, Anhui, People's Republic of China

* Corresponding author. Tel: +86 551 3492386, Fax: +86 551 3492386, Email: zjszyzzc{at}mail.hf.ah.cn


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Funding
 References
 
Aims: Heart failure is a life-threatening complication of high-dose cyclophosphamide (CTX) chemotherapy, and the present study aimed at identifying the mechanism involved in mice.

Methods and results: CTX at 800 mg/kg resulted in heart failure, in which cytoplasmic thioredoxin reductase (TrxR1) activity and non-protein free thiol (NPFT) level were suppressed by 90 and 62%, respectively. The combination of 350 mg/kg CTX and the glutathione synthesis inhibitor buthionine sulfoximine (BSO) also evoked heart failure, in which TrxR1 activity and NPFT level were suppressed by 66 and 62%, respectively. NPFT depletion alone by BSO did not cause cardiac toxicity. CTX at 350 mg/kg alone also did not cause cardiac toxicity, even though it suppressed TrxR1 activity by 68%. Previous studies have shown that half inactivation of TrxR1 in tumour, bladder, and kidneys was associated with toxicological consequences. Cardiac TrxR1 is dispensable, but cardiac cytoplasmic thioredoxin (Trx1) is essential. The potential uncoupling between TrxR1 and Trx1 may explain why there is no cardiac toxicity following TrxR1 inhibition. However, TrxR1 inactivation may still play a role in CTX-evoked heart failure because inactivated TrxR1 gains cytotoxic function, which may engender noticeable toxicity when massive NPFT is deleted.

Conclusion: CTX-evoked heart failure involves pronounced co-suppression of TrxR1 activity and NPFT level.

Key Words: Cyclophosphamide • Thioredoxin reductase • Non-protein free thiol • Cardiac toxicity

Received June 1, 2008; Revised July 2, 2008; Accepted November 3, 2008


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
The alkylating agent cyclophosphamide (CTX) is the most frequently used nitrogen mustard for antineoplastic chemotherapy and is an essential component of many effective drug combinations.1 High-dose CTX is the mainstay in most pre-transplant preparative regimens,2,3 and heart failure is a life-threatening complication of high-dose CTX chemotherapy.4,5 To date, little is known about the mechanism underlying CTX-related cardiac toxicity.

Mammalian thioredoxin reductase (TrxR) is an NADPH-dependent homodimer oxidoreductase with one flavin adenine dinucleotide and one selenocysteine per subunit.6 The major substrate of TrxR is oxidized thioredoxin (Trx). Moreover, TrxR can directly reduce many other endogenous and exogenous compounds such as lipid hydroperoxides and H2O2.7 There are three forms of mammalian TrxR isoenzymes—cytoplasmic TrxR (TrxR1), mitochondrial TrxR (TrxR2), and testis-specific TrxR (TGR)—all of which are selenoproteins.6 The Trx system plays an important role in the redox regulation of multiple intracellular processes, including DNA synthesis, transcriptional regulation, cell growth, and resistance to cytotoxic agents.8

TrxR is an excellent molecular target for anticancer agents.9 Prototype compounds representative of the major classes of clinically used anticancer alkylating agents effectively inhibit TrxR.10 We previously reported that CTX could powerfully inhibit TrxR1 activity in tumour.11 In addition, CTX also largely inhibits TrxR1 in bladder tissue, leading to severe haemorrhagic cystitis.12 On the basis of these observations, it is conceivable that CTX may cause cardiac TrxR1 inactivation, which may play a role in CTX-induced cardiac toxicity.

Among the compounds of non-protein free thiol (NPFT), glutathione (GSH) is the most abundant in mammalian cells with millimolar concentrations, and it plays a critical role in drug resistance.13 Depleting GSH at a level of 60% using the GSH synthesis inhibitor buthionine sulfoximine (BSO) does not cause cardiac toxicity,14 but largely sensitizes CTX to evoke cardiac deaths in mice or rats.15

In the present study, we investigated the impact of CTX on cardiac TrxR1 and NPFT levels.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Chemicals and drugs
NADPH, HEPES, insulin, 5,5'-dithiobis (2-nitrobenzotic acid) (DTNB), Trx (Escherichia coli), guanidine hydrochloride, GSH, bovine serum albumin, CTX, 1-chloro-2,4-dinitrobenzene (CDNB), and BSO were all purchased from Sigma (St Louis, MO, USA). Other chemicals were of the highest grade available.

Animals
Male Kunming mice (body weight of 20–22 g, 4–5 weeks) and male inbred line C57BL/6N mice (body weight 20–22 g, 6–7 weeks) were used in this study. The mice and their food were all purchased from Shanghai SLAC Laboratory Animal Co. Ltd, People's Republic of China. The mice were housed in plastic cages (6–8 mice per cage) in a room with controlled temperature (22 ± 1°C) and humidity (50 ± 10%) and 12 h light/dark cycle. The mice were allowed access to food and water ad libitum. All animal experiments were performed in strict compliance with the ethical guidelines issued by University of Science and Technology of China.

Animal treatments
Low-dose cyclophosphamide in Kunming mice
To investigate dose- and time-dependent changes of TrxR1 activity after CTX treatment, 42 male Kunming mice were randomly divided into six treatment groups with seven mice per group. The mice in groups I–IV were injected intraperitoneally (i.p.) with either saline (as the control), or 50, 150, or 250 mg/kg of CTX and were sacrificed 3 h after the injection. The mice in groups V and VI were injected i.p. with 250 mg/kg CTX and were sacrificed at 30 and 120 h after CTX administration, respectively.

Low-dose cyclophosphamide in C57BL/6N mice with tumours
To observe recovery in TrxR1 activity following inhibition and to compare the restoration of TrxR1 activity between heart and tumour tissues, 20 male C57BL/6N mice were implanted with Lewis lung carcinoma (LLC) cells. When the tumour had grown to ~500 mm3, the mice were randomly divided into four treatment groups with five mice per group. Group I was treated with saline and acted as the control. Groups II–IV were injected i.p. with a single dose of CTX (250 mg/kg). Animals from groups II to IV were sacrificed at 3, 30, and 120 h, respectively, after CTX administration, and animals from group I were sacrificed at 3 h after saline administration.

Low-dose cyclophosphamide and buthionine sulfoximine combination in Kunming mice
Male Kunming mice were randomly divided into seven treatment groups with five mice per treatment. Control: the mice were injected i.p. with saline and sacrificed 3 h after treatment. CTX alone (3 or 18 h): the mice were injected i.p. with 350 mg/kg CTX and sacrificed at 3 or 18 h after CTX administration. BSO alone (3 or 18 h): BSO was given by i.p. injection at 1000 mg/kg twice with a 12 h interval between doses, the mice were sacrificed at 3 and 18 h after the second dose of BSO had been administered for 4 h. BSO and CTX combination (3 or 18 h): the procedure of BSO administration was exactly the same as the BSO alone, 4 h after the second BSO administration, the mice were injected i.p. with 350 mg/kg CTX and sacrificed at 3 or 18 h after CTX administration.

High-dose cyclophosphamide and buthionine sulfoximine combination in Kunming mice
Five male Kunming mice were treated with 550 mg/kg CTX with prior administration of BSO. The BSO regimen was identical to that used in the low-dose CTX and BSO combination experiment. Mice were monitored for survival. For studies of the changes in TrxR1 activity and NPFT level, 10 male Kunming mice were randomly divided into two groups with five mice per group. One group was the control, and the other group received 550 mg/kg CTX and BSO combination. All animals were sacrificed at 4 h after CTX administration.

High-dose cyclophosphamide in Kunming mice
Male Kunming mice were randomly divided into three groups with six mice per group. Group I was treated with saline as the control, groups II and III were i.p. injected with a single dose of CTX (800 mg/kg). Animals in groups II and III were sacrificed at 3 and 18 h, respectively, after CTX administration, and animals in group I were sacrificed at 3 h after saline administration.

Sample preparation
At the end of each set of experiments, the mice were sacrificed by cervical dislocation. Peripheral blood collected from ophthalmic veins was centrifuged to obtain serum. The hearts and tumours were excised and rinsed in ice cold saline. The samples were stored at –30°C before assay.

Biochemical parameters
The activity of creatine kinase (CK), lactate dehydrogenase (LDH), and aspartate aminotransferase (AST) in serum was estimated using commercial kits.

Hearts and tumours were homogenized with ice cold 150 mM NaCl (1:9 w/v). For NPFT assay, immediately after homogenization, a volume of homogenate was removed and mixed with trichloroacetic acid (20%w/v) to precipitate protein, at a ratio of 10:1 in volume and then centrifuged at 10 000g and 4°C for 5 min. Within 2 h after the centrifugation, the resulting supernatant was mixed with DTNB and read at 412 nm.16 The amount of NPFT was determined by using GSH as standard and expressed as nmol thiol/mg protein. For antioxidant enzyme assays, the rest of the homogenate was centrifuged at 15 000g and 4°C for 15 min. The resulting supernatants were used for the determination of TrxR1, GPx, GST, catalase (CAT), and total superoxide dismutase (SOD) activity. Protein levels were determined by Bradford dye-binding assay with bovine serum albumin as the standard.

TrxR1 activity was measured based on the method of Holmgren and Bjornstedt,17 with some modifications as described previously.18 The stock mixture contained HEPES buffer (0.25 M), NADPH (2.5 mM), EDTA (10 mM), and insulin (1 mM), with a final pH of 7.6. In a 96-well plate, 7 µL stock mixture, 3 µL Trx (0.17 mM), 40 µL HEPES (50 mM, pH 7.6), and 10 µL sample (with 40~60 µg protein) were added into a well. The enzymatic reaction was maintained at 37°C for 20 min and then was stopped by adding 240 µL terminative solution (0.5 mM DTNB/6 M guanidine hydrochloride in 0.2 M Tris–HCl, pH 8.0). Each sample contained a non-enzymatic reaction in which Trx was substituted by saline, but other components were exactly the same as the enzymatic reaction. The 96-well plates were read at 412 nm. The A412 increase was calculated by subtracting the absorbance of the non-enzymatic reaction from the absorbance of the enzymatic reaction. A background control, which was the subtraction of absorbance with and without Trx in the absence of sample, was further subtracted from the A412 increase. According to the standard curve of TrxR1 obtained with mouse heart, 70 µg protein was in the linear range of detection (correlation coefficient R2 = 0.998; data not shown). The TrxR1 activity unit was defined as A412 changex1000/min19 and was expressed as U/mg protein. GPx was assayed by the method of Rotruck et al.20 One unit of GPx activity was calculated in terms of µmol of GSH oxidized/min. GST activity was chemically determined using CDNB. One unit of GST activity was calculated in terms of nmol CDNB changed/min/mg of tissue protein.21 CAT activity was assayed on the basis of its ability to decompose H2O2 and measured at 240 nm.22 One unit of CAT activity was defined in terms of nmol H2O2 consumed/min/mg of tissue protein. Total SOD activity was assayed by using the system of xanthine–xanthine oxidase and nitroblue tetrazolium (NBT). One unit of SOD activity was defined as the amount of protein that inhibits the rate of NBT reduction by 50%. Data are expressed as U/mg of tissue protein.23

Histopathological observation
Heart tissues were immediately excised after sacrifice and rinsed with ice cold saline. Then they were fixed in 10% neutral-buffered formalin solution and embedded into molten paraffin wax. Tissue sections of 5 µm thickness were stained with haematoxylin and eosin (H&E) and observed under a light microscope by an experienced pathologist who was blinded to the treatments.

Statistical analysis
Data are presented as mean ± SD. Statistical significance was determined by one-way analysis of variance followed by Tukey's multiple comparison. A P-value of less than 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
Cyclophosphamide preferentially suppresses cardiac TrxR1 activity over other antioxidant parameters
Three hours after administration of a bolus dose of CTX (50 and 150 mg/kg) to Kunming mice, TrxR1 activity was significantly decreased in a dose-dependent manner, by 19 and 57%, respectively, compared with control. At a CTX dose of 250 mg/kg, 62% inhibition was observed compared with control; however, this was not significant (P > 0.05) when compared with the 150 mg/kg dose (Figure 1A).


Figure 1
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  		Figure 1 Effects of cyclophosphamide (CTX) on thioredoxin reductase (TrxR1) activity and other redox parameters in heart. (A) Different doses of CTX were injected i.p. into Kunming mice and TrxR1 activity in the heart was tested at 3 h. P-value indicates the comparison with control. (B) Changes in other tested antioxidant parameters. Changes are given as the percentage of the control value. The control activity of GPx, GST, SOD, CAT, and TrxR1 was 80.1 ± 8.3, 203.1 ± 13.8, 1.2 ± 0.1, 1191.6 ± 203.2, and 110.4 ± 9.6 U/mg protein, respectively, and the control NPFT level was 26.5 ± 1.1 nmol thiol/mg protein. a, P < 0.001 when compared with the corresponding control. Data are presented as mean ± SD (n = 7).

 
In parallel to TrxR1, after 250 mg/kg CTX treatment for 3 h, the activities of antioxidant enzymes (GPx, GST, CAT, and SOD), as well as NPFT levels, were also determined. The NPFT level was slightly but significantly decreased by 17% (P < 0.001; Figure 1B), whereas other antioxidant parameters tested showed no significant variation (Figure 1B). Together, these data demonstrate that CTX preferentially suppresses cardiac TrxR1 activity over other antioxidant parameters, including the NPFT level.

Cardiac TrxR1 activity recovers less efficiently
After treatment with 250 mg/kg of CTX, TrxR1 activity decreased to a nadir at 3 h and then gradually recovered over a 120 h time frame (Figure 2A). We previously reported that at 3 h after CTX treatment, TrxR1 activity in bladder and tumour considerably decreased, but was fully restored to normal levels within 24 and 120 h, respectively.11,12 In contrast to the full restoration in tumour and bladder, cardiac TrxR1 recovery appeared less efficient because TrxR1 activity at 120 h was still significantly lower than the control (P < 0.01; Figure 2A). To confirm that this is a unique profile of cardiac TrxR1, inbred line C57BL/6N mice-bearing LLC were i.p. injected with 250 mg/kg CTX. TrxR1 activity in the tumour tissue rapidly recovered, but in the heart it did not recover (Figure 2B).


Figure 2
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  		Figure 2 Time course of thioredoxin reductase (TrxR1) activity in heart and tumour. Kunming mice and C57BL/6N mice-bearing Lewis lung carcinoma were i.p. injected with 250 mg/kg cyclophosphamide, and TrxR1 activity in heart and tumour was tested at a designed time. (A) TrxR1 activity in heart of Kunming mice. All bars represent mean±SD (n = 7). (B) TrxR1 activity in heart and tumour of C57BL/6N mice. Data are presented as mean±SD (n = 5). P-value indicates the comparison with the corresponding control.

 
In these time course experiments, we also determined the activities of serum CK, LDH, and AST, which are well-known diagnostic indicators of cardiac injury. CTX at 250 mg/kg did not cause a significant increase in any of these enzyme activities during the experimental period (data not shown), confirming the theory that the risk of cardiac toxicity of CTX is only associated with high doses.

TrxR1 and non-protein free thiol changes in a model of heart failure-related death caused by cyclophosphamide and buthionine sulfoximine combination
Friedman et al.15 demonstrated that administration of CTX to mice resulted in sudden death within 3 h that had been pre-treated with BSO. Subsequently, they found that administration of CTX to rats pre-treated with BSO also caused 100% death within 4 h. By monitoring the heart rate, blood pressure, and electrocardiogram of the rat hearts, they revealed that the major cause of acute death was heart failure. In this study, we used this heart failure-related model to investigate the biological changes in the heart. According to Friedman et al., 500 mg/kg CTX in animals is equivalent to a dose of 240 mg/kg in humans, which can cause cardiac damage. In our experiments, we used 350 and 550 mg/kg CTX; thus our doses are relevant to clinical usage. In the first experiment, BSO plus 550 mg/kg CTX resulted in 100% death during 4–6 h, and cardiac TrxR1 activity and the NPFT level decreased by 77 and 63%, respectively, at 4 h (Figure 3A and B). These results show that pronounced co-suppression of cardiac TrxR1 activity and NPFT level is involved in CTX-evoked heart failure. In the second experiment, mice treated with BSO plus 350 mg/kg CTX could survive to 18 h. At 3 h, CTX alone significantly decreased cardiac TrxR1 activity by 68% and NPFT level by 18% (Figure 4A and B); BSO alone significantly depleted cardiac NPFT by 46% (Figure 4B). Only the combined treatment pronouncedly co-suppressed cardiac TrxR1 activity by 66% and NPFT level by 62% (Figure 4A and B). Consistent with the conclusion drawn from the first experiment, i.e. pronounced co-suppression of cardiac TrxR1 activity and NPFT level is involved in CTX-evoked heart failure, we found that CTX and BSO together at 18 h caused cytoplasmic vacuolization, dissolution of myocardial fibres, myofilament loss (Figure 5D), and a massive increase in serum CK, LDH, and ASK activities (Figure 6). Whereas neither CTX nor BSO alone caused pronounced co-suppression of TrxR1 and NPFT, accordingly, they did not induce cardiac toxicity (Figures 5B and C and 6).


Figure 3
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  		Figure 3 Effect of high-dose cyclophosphamide and buthionine sulfoximine on cardiac thioredoxin reductase (TrxR1) activity and non-protein free thiol (NPFT) level. (A) TrxR1 activity and (B) NPFT level. Data are presented as mean±SD (n = 5). a, P < 0.001 when compared with control.

 


Figure 4
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  		Figure 4 Effect of low-dose cyclophosphamide and buthionine sulfoximine on cardiac thioredoxin reductase (TrxR1) activity and non-protein free thiol (NPFT) level. (A) TrxR1 activity and (B) NPFT level. Data are presented as mean±SD (n = 5). a, P < 0.001 when compared with control.

 


Figure 5
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  		Figure 5 Effect of low-dose cyclophosphamide (CTX) and buthionine sulfoximine (BSO) on cardiac architecture. (A) Control and (B) 350 mg/kg CTX alone. (C) BSO alone. (D) The combination of BSO and CTX. Solid arrows indicate myocardial fibres dissolving with cytoplasmic vacuolization; open arrows indicate myofilament loss with cytoplasmic vacuolization.

 


Figure 6
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  		Figure 6 Effect of low-dose cyclophosphamide and buthionine sulfoximine on serum creatine kinase (CK), lactate dehydrogenase (LDH), and aspartate aminotransferase activities. At 18 h, CK, LDH, and AST activities in serum were measured. Changes are given as the fold of the control value, which was the activity of the control sacrificed at 3 h. The control activity of CK, LDH, and AST was 58.5 ± 11.4, 34.6 ± 3.3, and 37.5 ± 4.9 U/mL, respectively. Data are presented as mean±SD (n = 5). P-value indicates comparison with the corresponding control.

 
High-dose cyclophosphamide itself pronouncedly co-suppresses cardiac TrxR1 activity and non-protein free thiol level
After administration of 800 mg/kg of CTX, cardiac TrxR1 activity was drastically inhibited by 90% and NPFT was markedly depleted by 62% at 3 h, and these changes were maintained at 18 h (Figure 7A and B). Moreover, we found the activity of CK, LDH, and AST tremendously increased in serum (Figure 8).


Figure 7
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  		Figure 7 Effect of high-dose cyclophosphamide (CTX) on cardiac thioredoxin reductase (TrxR1) activity and non-protein free thiol (NPFT) level. Mice were injected i.p. with 800 mg/kg CTX. TrxR1 activity and NPFT level in the heart were tested at 3 and18 h after the treatment. (A) TrxR1 activity and (B) NPFT level. Data are presented as mean±SD (n = 6). P-value indicates comparison with the corresponding control.

 


Figure 8
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  		Figure 8 Changes of serum creatine kinase (CK), lactate dehydrogenase (LDH), and aspartate aminotransferase activities after cyclophosphamide (CTX) administration. Mice were injected i.p. with 800 mg/kg CTX, and CK, LDH, and AST activities in serum were measured at 18 h after the treatment. Changes are given as the fold of the control value. The control activity of CK, LDH, and AST was 50.1 ± 3.0, 38.6 ± 2.4, and 29.0 ± 7.6 U/mL, respectively. Data are presented as mean±SD (n = 6). P-value indicates comparison with the corresponding control.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
TrxR has a uniquely accessible selenocysteine residue compared with many other selenoproteins and therefore is more susceptible to inactivation by alkylating agents than other selenoproteins or thiol-containing cellular scavengers such as GSH.10 Our results are consistent with these findings; 250 mg/kg CTX prominently suppressed cardiac TrxR1 activity (Figure 1A), whereas GPx activity was not affected (Figure 1B), the suppression of CTX on NPFT level was less pronounced when compared with TrxR1 activity (Figure 1B).

The preferential suppression of CTX on TrxR1 over NPFT and other antioxidant enzymes in heart is identical with what we observed in tumour11 and is similar to the action of ifosfamide in kidney.18 However, cardiac TrxR1 appears unique in two aspects compared with its counterparts in other tissues. First, TrxR1 inactivation by half due to CTX or ifosfamide treatment is associated with a functional consequence, such as deceleration of ascitic cancer cell proliferation,11 haemorrhagic cystitis,12 and nephrotoxicity.18 In contrast, when cardiac TrxR1 activity was suppressed by 68%, no cardiac toxicity was observed. Secondly, TrxR1 activity in tumour tissue, ascitic cancer cells, bladder, or kidney rapidly recovers to normal levels after being inactivated by CTX or ifosfamide, but in heart, the recovery was slower in Kunming mice (Figure 2A) and could not even be observed in inbred line C57BL/6N mice (Figure 2B). These phenomena confirm the theory that cardiac TrxR1 is essential in most tissues except the heart.24 TrxR1 is expressed in cells with a strong proliferative activity, such as neoplastic tissues and tumour cell lines,25,26 and it has also been shown to be a c-Myc target in a human B-cell line,27 indicating a role of TrxR1 in cell proliferation. The myocardium consists of cells that have limited regenerative capability.28 Thus, the unique characteristics of cardiac TrxR1 may correlate with the proliferative profile of cardiac cells.

The notion that cardiac TrxR1 is dispensable appears contradictory to the findings that cytoplasmic Trx (Trx1) plays an important role in the heart.29,30 The heart-specific over-expression of dominant-negative Trx1 is associated with increased oxidative stress and cardiac hypertrophy in mice, whereas over-expression of wild-type Trx1 is beneficial.29 These findings imply that some important functions of cardiac Trx1 may not depend on TrxR1;24 accordingly, the suppression of TrxR1 by CTX does not definitely matter overall dysfunction of the Trx1 pathway. The potential uncoupling between cardiac TrxR1 and Trx1 may explain why the heart is distinct when TrxR1 activity is inhibited by CTX. In tumour tissue, ascitic cancer cells, bladder, and kidney, TrxR1 is coupled with Trx1, TrxR1 inhibition by half leads to dysfunction of the Trx1 system, and generates an immediate toxicological consequence. In the heart, after TrxR1 is inhibited, Trx1 may still work efficiently due to this potential uncoupling;24 thus there is no measurable cardiac toxicity, which is corroborated by using the TrxR1-specific inhibitor auranofin. Auranofin at 100 mg/kg inhibits cardiac TrxR1 activity by 65% in rats, but has no effect on initial heart function in relation to coronary flow, heart rate, and developed pressure.31

The inhibition of the disulphide-reducing activity of TrxR1 by CDNB or curcumin forms selenium-compromised TrxR1 with NADPH oxidase activity, leading to the accumulation of reactive oxygen species.32,33 The induction of cell death was promoted by selenium-compromised TrxR1 with or without NADPH oxidase activity.34 Selenium-compromised TrxR1 could act as a cell killer by inducing apoptotic cell death, thus it was designated as selenium-compromised thioredoxin reductase-derived apoptotic protein (SecTRAP).35 The potential uncoupling between cardiac TrxR1 and Trx1 does not exclude that TrxR1 inactivation still plays a role in CTX-evoked heart failure, because inactivated TrxR1 (SecTRAP1) has cytotoxic activity, which could emerge under certain circumstances such as NPFT depletion. CTX at 350 mg/kg suppressed cardiac TrxR1 by 68% (Figure 4A); BSO suppressed cardiac NPFT by 46% (Figure 4B), but no cardiac toxicity was observed in either single treatment. The combination of CTX and BSO suppressed cardiac TrxR1 activity by 66% and NPFT level by 62% (Figure 4A and B), which is a similar extent to that seen with the single treatment; however, severe cardiac toxicity was observed. These results suggest that the cytotoxic effect of SecTRAP1 becomes apparent in an environment with largely compromised NPFT. In this regard, the ceiling dose of CTX (800 mg/kg) provides further evidence as it caused catastrophic heart failure in which SecTRAP1 co-exists with largely compromised NPFT. As the principal NPFT, GSH plays a critical role in drug resistance.13 GSH depletion by BSO greatly increased the cytotoxic effect of arsenic trioxide, an inhibitor of TrxR. The mechanism was considered to be dysfunction of both GSH and Trx systems.36 Furthermore, the mechanism probably also includes cytotoxic activity of SecTRAP1 when the GSH level is lower.

In the present study, we observed that TrxR1 activity decreased in a dose-dependent manner when CTX doses were within 150 mg/kg (Figure 1A), and cardiac TrxR1 activity recovered less efficiently after being inhibited by CTX when compared with other tissues (Figure 2). Thus, there may be a cumulative inhibition of TrxR1 activity in the heart after repeated exposure to low-dose CTX, suggesting that repeated low-dose exposure could produce a comparable extent of TrxR1 inhibition as seen from single administration of high-dose CTX. Whether repeated low-dose exposure causes heart failure or not would depend on cardiac NPFT levels, and it is speculated that individuals with lower levels of NPFT in the heart are at high risk of heart failure.

Interestingly, we also observed recovery of TrxR1 activity in the hearts of Kunming mice within 120 h (Figure 2A). It is well known that oxidative stress activates the transcriptional factor NF-E2-related factor 2 (Nrf2), which binds to the promoter containing antioxidant-responsive element (ARE). The TrxR1 gene possesses ARE and thus TrxR1 transcription increases under oxidative stress. Sakurai et al.37 reported that the TrxR1 gene was induced under cadmium-induced oxidative stress through Nrf2 activation and binding to ARE in the TrxR1 gene promoter. Chen et al.38 reported that HNE, at sublethal concentrations, induced an adaptive response and enhanced cell tolerance, primarily through induction of TrxR1 via transcriptional activation of the Nrf2 signalling pathway. Therefore, we infer that the Nrf2-ARE-TrxR transcription pathway might be a major mechanism for the observed recovery of TrxR1 activity in CTX-treated hearts.

Unlike TrxR1, heart-specific inactivation of TrxR2 leads to fatal dilated cardiomyopathy; thus, TrxR2 is essential in heart function.39 We did not present data on TrxR2 because its activity could not be assessed. Basically, the standard TrxR activity assay17 determines insulin sulfhydryl by using DTNB at A412 nm and relies on the difference between an enzymatic reaction in the presence of Trx and a non-enzymatic reaction in the absence of Trx. The background sulfhydryl of non-enzymatic reaction contributed by either protein or non-protein has a pivotal impact on the assay. When the value of the non-enzymatic reaction is low and the TrxR activity is high, the difference between the enzymatic and non-enzymatic reactions is far away from the value of non-enzymatic reaction, and thus the assay method is feasible; this is the case for the cardiac TrxR1 assay. Conversely, when the value of the non-enzymatic reaction is high and the TrxR activity is low, the value of the enzymatic reaction is close to that of the non-enzymatic reaction, thus resulting in an unacceptable assay precision; this is the case for the cardiac TrxR2 assay. Modifications in an attempt to reduce background interference or to increase the value of the enzymatic reaction have been reported, for example, heating tissue homogenate at 50°C for 10 min to precipitate some heat-unstable protein and thus to reduce protein sulfhydryl, dialysing tissue homogenate to reduce non-protein sulfhydryl, and increasing the amount of Trx to enhance the value of enzymatic reaction.19 For the extract of cardiac mitochondria, we tried using all of the above-mentioned protocols, but no satisfactory improvement in the increase of A412 nm could be achieved, probably due to a very low TrxR2 activity therein. To the best of our knowledge, cardiac tissue TrxR2 activity measured by insulin/Trx has never been reported previously. As the catalytic centres of TrxR1 and TrxR2 are virtually identical, Jakupoglu et al.24 speculated that it would be extremely difficult to design chemical drugs with specificity for TrxR1. It is conceivable that CTX also attacks cardiac TrxR2, but to what degree cannot be predicted due to organelle compartment and the limitation of the assay method. Thus, at the present stage with the absence of information about TrxR2, we conclude that CTX-evoked heart failure involves pronounced co-suppression of cardiac TrxR1 activity and NPFT level.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
This work was supported by a grant from University of Science and Technology of China (KY2002).


   Acknowledgement
 
The authors would like to acknowledge Dungeng Peng and Huali Wang, as well as Hongjuan Lu for technical assistance.

Conflict of interest: none declared.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
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 References
 

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