Occult Cardiotoxicity: Subtoxic Dosage of Bis(2-chloroethoxy)methane Impairs Cardiac Response to Simulated Ischemic Injury

Abstract

The effect of Bis(2-chloroethoxy)methane (CEM) on myocardial response to ischemia was tested in rats. CEM was dermally applied for 3 days to F344/N male rats, at 0, 100, 400, or 600 mg/kg/d. Subsequently, left ventricular sections were prepared from each rat heart. Part of the sections from each heart were exposed to 90 minutes of simulated ischemia, followed by 90 minutes of reoxygenation. The rest of the sections were continuously oxygenated. Mitochondrial activity was assessed in the sections by the MTT colorimetric assay, reflecting dehydrogenases redox activity. Myocardial toxicity occurred in response to 400 and 600 mg/kg, characterized by myofiber vacuoles, necrosis, and mononuclear infiltrates. The latter dose was lethal. In sections from rats treated with 400 mg/kg CEM, redox activity was decreased by 21% (p < 0.01) in oxygenated conditions and by 45% (p < 0.01) in ischemia-reoxygenation, compared to controls. Hearts of rats treated with 100 mg/kg/d CEM showed normal histology. Their mitochondrial activity did not differ from that of untreated rat hearts in oxygenated conditions. However, in ischemia-reoxygenation, their redox activity was significantly lower (by 46%, p < 0.01) than that of untreated rat hearts. These results demonstrate that subtoxic dosage of a cardiotoxic agent may cause occult cardiotoxicity, reflected by impaired response to ischemia.

Introduction

Environmental chemical exposure may contribute to heart disease, and account, at least partially, for the variability among different individuals in their response to cardiac ischemic injury. Bis(2-chloroethoxy)methane (CEM), an organic solvent used as an intermediate for polysulfide rubber, was found to exert cardiotoxic effects in rodents, characterized by myocyte cytoplasmic vacuolation due to mitochondrial swelling, focal necrosis, and inflammatory infiltrates (Dunnick et al., 2004a). Ultrastructural studies showed that CEM induces mitochondrial swelling with disruptions of the cristae and the mitochondrial membrane, pointing to the mitochondria as the primary site for CEM-induced cardiotoxicity (Dunnick et al., 2004b). This finding was recently supported by a thorough analysis of the changes in cardiac gene expression patterns in rats in response to the dermal application of CEM. This analysis revealed that changes in the expression of genes involved in energy metabolism are most prominent in the cardiac response to exposure to CEM (Dunnick et al., 2006).

The cardiotoxic effect of CEM is dose-dependent, and has been described in response to doses of 200 mg/kg or higher. Doses of 600 mg/kg/d exert dramatic cardiotoxic effects, and are lethal in a significant percentage of rats receiving it. (Dunnick et al., 2004a, 2004b). Lower doses than 200 mg/kg/d do not exert significant morphological effects on the heart (Dunnick et al., 2004a). The effect of the dose of 200 mg/kg on the expression of genes involved in energy metabolism was very moderate (Dunnick et al., 2006). The half-life of CEM in the rat heart is approximately 30–40 minutes (NTP, 2006).

Damage to cardiac mitochondria would be expected not only to cause a direct effect on myocyte viability, but also on ability to withstand ischemic conditions. To the best of our knowledge, the effect of CEM on the cardiac response to ischemia has not been tested. The assessment of 3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction activity is extensively used as a convenient colorimetric agent for measuring cytotoxicity and cytoprotection in different cell types, including cardiomyocytes (Mojzisova et al., 2006).

In whole cells, MTT reduction to a formazan product is an indicator of the cell pyridine nucleotide redox state, providing readout of the cellular redox activity (Shearman et al., 1994). In the heart, MTT reduction serves as an indicator of mitochondrial function, and reflects cardiac energy metabolism levels (Kakinuma et al., 2000; Bes et al., 2004). The goal of the present study was to assess the cardiotoxic effect of CEM in conditions of exposure to high oxygen levels and to simulated ischemia/reperfusion, using the MTT colorimetric assay.

Materials and Methods

Materials

CEM (CAS # 111-91-1, MW = 173.04), 98.5% pure, was obtained from Karl industries, Aurora, OH, USA. The chemical was prepared for dosing as previously described by Dunnick et al. (2004a). The 3-[4,5 dimethylthiazol-2yl]-2.5 diphenyltetrazolium bromide (MTT) was obtained from Sigma (St. Louis, MO, USA).

The incubation medium for ex vivo experiments, glucose–phosphate-buffered saline (G-PBS) was prepared daily in distilled water. The buffer consisted of (in mM): NaCl 136.9, KCl 2.68, Na₂HPO₄ 8.10, KH₂PO₄ 1.53, MgCl₂·6H₂O 0.5, CaCl₂·2H₂O 0.9, glucose 5.55 (pH 7.45).

MTT solution was prepared by dissolving 0.5 mg of MTT in 1 ml of G-PBS.

Animals

Female F344 rats (Harlan Laboratories, Jerusalem, Israel), 5–6 weeks old, weighing 90–100 g were used in this study. Animal handling was conducted in accordance with the NIH guidelines (Grossblatt, 1996). The study was approved by the Institutional Ethical Committee for Animal Research of Hadassah Medical Center and the Hebrew University, Jerusalem, Israel.

Dermal Administration of Cem and Specimen Acquisition

Rats were anesthetized with Ketamine/Xylazine. The fur of the upper dorsal interscapular area was clipped. Adjustable micropipettes with disposable tips were used for CEM application. The substance was applied daily onto the shaved skin at doses of: 0 (n = 7), 100 (n = 10), 400 (n = 9), or 600 mg/kg (n = 9) for 3 consecutive days. For the second and third application of CEM the rats were kept in a restriction cage, and no anesthesia was used. Four hours after the final application of CEM the animals were injected with sodium heparin (250 U intraperitoneally) and 30 minutes later were anesthetized. The hearts were immediately removed and placed in heparinized ice-cold saline solution.

The hearts were cut longitudinally, and a 3-mm-thick section was fixed in buffered formalin for histopathological assessment, performed on standard hematoxylin and eosin stained 5-μm sections. The histopathological examination was conducted in a blinded manner. The examining pathologist was not aware of the treatment and dosage given to the rats. The left ventricular tissue of the rest of the heart was separated, and cut manually into small cubes of ~2 mm for ex vivo experiments.

Ex Vivo Experiments

The use of rat ventricular sections in an ex vivo experimental system simulating ischemia and reoxygenation (I/R) was modified from our earlier studies in isolated human right atrial sections (Schneider et al., 2003; Ad et al., 2005). The simulated ischemia-reoxygenation exposure consisted of 30 minutes of equilibration, 90 minutes of simulated ischemia, and 90 minutes of reoxygenation. Specifically, left ventricular cubes from the mid-section of the left ventricle, ~2 mm, were exposed to 30 minutes of aerobic equilibration in 25 ml of G-PBS, bubbled with oxygen at 37°C (O₂-G-PBS), then washed in PBS and transferred for 90 min to conditions of simulated ischemia (N₂ –PBS, at 37°C), and then to 90 minutes of reoxygenation (O₂-G-PBS, 37°C), in the presence of MTT. Aerobic control sections from each heart were incubated in O₂-G-PBS throughout the entire experiment. The protocol of the ex vivo experiment is illustrated in Figure 1.

It should be emphasized, that test tubes of the simulated ischemic conditions were vigorously flushed with 100% N₂ for 5 minutes in order to replace the oxygen. En route to the tubes, the gas passed through 2 traps, the first containing 1% Na₂SO₃, and the second containing water. The flow of N₂ was attenuated to ~1 bubble/sec at the exit trap. Figure 2 illustrates the system of the ex vivo experiment of simulated ischemia.

MTT Assay

At the end of each experimental protocol, the reduction of 3-[4,5 dimethylthiazol-2yl]-2.5 diphenyltetrazolium bromide (MTT) to blue formazan by mitochondrial dehydroge-nases in the tissue was assessed.

The sections were exposed to MTT for the entire period of reoxygenation—5 sections were incubated in 20 ml O₂-G-PBS containing MTT (0.5 mg/ml) at 37°C for 90 minutes. The sections were then transferred to a small test tube containing 3 ml saline, which was shaken for 1 minute to remove excess dye. Then, the sections were wiped on gauze cloth and transferred to 15 ml plastic test tubes and frozen overnight. Extraction of the Formazan dye into 10 ml of dimethyl sulfoxide (DMSO) was done with vigorous shaking for 1 hour at 37°C. The absorbance of the colored supernatant was measured spectrophotometrically at 500 nm. The sections were dried in a 90°C oven for 24 hours and weighed.

The results are expressed as optical density (OD)/mg dry weight of myocardial tissue. The effect of ischemia is derived from the ratio between the I/R optical density to that of its aerobic controls, normalized to dry weight of myocardial tissue.

Statistical Analysis

Results are expressed as Mean ± SEM. Statistical significance of differences between groups was carried out using ANOVA and Tukey’s post-hoc tests. Statistical differences of p < 0.05 were considered significant.

Results

Body Weight, Mortality and Clinical Signs

Mean body weight of CEM-treated rats did not differ from that of controls. Two of the first three animals that received 600 mg/kg/d CEM died after the second application of CEM, and the third showed severe dyspnea. Due to the early death in this group, it was decided to sacrifice the animals and cease further evaluation of their heart in ex vivo experiments. Rats that received 100 and 400 mg/kg/d CEM lived throughout the 3 day experiment and did not show clinical signs of toxicity.

Histopathology

Rats that received 100 mg/kg CEM (n = 10) showed no histological abnormalities of the heart, and did not differ from control, untreated rats (n = 7). The histopathological findings in rats that received 400 mg/kg CEM (n = 9) are presented in Figure 3. Pathological findings in these hearts include myofiber vacuolation (degeneration), myofiber fragmentation (necrosis), and inflammation. The vacuolation results from mitochondrial as well as ER distension, and is probably related to cytoplasmic myofibrillar loss following the primary mitochondrial damage (Dunnick et al., 2004b).

Similar changes, but more severe, were encountered in the rats that received 600 mg/kg CEM. The examining pathologist (A.N.), who examined the histological slides in a blinded manner, identified all the specimens of rats that received CEM 400 or 600 mg/kg/d, and could differentiate between the doses. Specimens of rats that received CEM 100 mg/kg/d could not be differentiated from these of untreated controls.

Ex Vivo Experiments

When the left ventricular sections were exposed to a sufficient supply of oxygen and glucose (O₂-G-PBS only), sections from rats treated with 100 mg/kg/d CEM did not differ from those from untreated rats as regards MTT reduction. However, sections from rats treated with 400 mg/kg/d CEM showed decreased MTT reduction, by 21% (p < 0.01), compared to sections from untreated rats (Figure 4).

Under conditions of simulated ischemia and reoxygenation, sections from rats treated with either 100 mg or 400 mg CEM showed severe impairment in MTT reduction.

They showed decreased MTT reduction by 46% and 45% (p < 0.01), compared to sections from untreated rats, that were exposed to the same conditions of simulated ischemia-reoxygenation (Figure 5).

The ratio between MTT reduction in ischemic and fully oxygenated conditions (MTT recovery, Ad et al., 2005), which reflects the metabolic reserves enabling the cell to withstand ischemic injury, was obviously significantly lower in CEM-treated rats, both at 100 and 400 mg/kg/d, than in untreated rats (Figure 6).

Discussion

The most significant finding of the present study is the occult cardiotoxic effect of a low dose of CEM, 100 mg/kg/d. The effect of this dose is not apparent as long as the tissue receives adequate supplies of glucose and oxygen. Cardiac morphology is normal, and MTT-reduction in O₂-G-PBS is not different from that of untreated rats. However, when the heart tissue is exposed to an injurious ischemic stimulus, simulated by the exposure to N₂-PBS, its metabolic reserves do not suffice, and it shows a dramatically decreased level of MTT-reduction compared to tissue from untreated rats. In other words, the occult cardiotoxic effect of 100 mg/kg/d CEM is demonstrable only when the heart tissue is exposed to a metabolic challenge. Exposure of the tissue to a high dose of CEM, 400 mg/kg, that is overtly cardiotoxic by morphologic criteria (Dunnick et al., 2004a), is associated with a decrease in MTT-reduction also in the presence of high glucose and oxygen levels. The tissue damage induced by 400 mg/kg/d CEM is further accentuated when the tissue is exposed to ischemic conditions.

The overall effect of the CEM showed a clear dose-response relationship: the effect of 600 mg/kg was deleterious, 400 mg/Kg caused obvious morphologic and biochemical toxicity, and 100 mg/kg caused only occult toxicity. However, it is noteworthy that under the conditions of simulated ischemia, there was no difference in the response to the CEM doses of 100 and 400 mg/kg. To account for this discrepancy, we suggest that ischemia accentuates the toxic effect of CEM. Thus, the dose-response curve is dramatically shifted to the left, and both 100 and 400 mg/kg are on the plateau of the maximal effect of CEM under these conditions. Alternatively, it could be argued that in response to the potentially lethal combination of CEM and ischemia, compensatory mechanisms are induced that maintain the cell viability, and offset the difference between the doses. Such mechanisms could be elicited by the acute exposure of the energy-depleted myocardium to ischemia, or by the repeated exposure to CEM, in the form of “chemical preconditioning,” similar to that elicited by cyclosporin A (Schneider et al., 2003). Such preconditioning could also account for the healing of myocardial tissue after a prolonged repeated exposure to CEM (Dunnick et al., 2006).

The previous finding by Dunnick et al. (2004b), locating the CEM-induced injury primarily at the mitochondria, is consistent with the present results. Mild damage to the mitochondrion would be expected to decrease cellular energy reserves, rendering the cell more susceptible to ischemia. The intensity of cellular damage due to ischemia depends not only on the magnitude and duration of the ischemic stimulus, but also on the type and metabolic status of the host cell, and on the status and inducibility of the immune system (Kumar et al., 2005). Many factors, genetic and environmental, including some toxicants, are likely to affect the metabolic status of cardiomyocytes, thereby determine their susceptibility to ischemia.

Different conditions are known to affect the susceptibility of cardiac myocytes to ischemia. The most widely studied of these is preconditioning: repeated short ischemic events render the myocyte less susceptible to prolonged ischemia. This relative tolerance to ischemia is reflected by improved MTT reduction under conditions of simulated ischemia (Ghosh et al., 2000). Similar effects of drugs are referred to as chemical (or pharmacologic) preconditioning (Loubani and Galinanes, 2002). Mitochondrial function has been shown to play a major role in the phenomenon of preconditioning (Hassouna et al., 2006). It is therefore conceivable that other chemicals affecting the mitochondrion, such as CEM, have an opposite effect of occult cardiotoxicity, or a dual effect of both toxicity and preconditioning protection.

Arrhythmias in response to ischemic events cause a significant number of cardiac deaths. Therefore, susceptibility to arrhythmias constitutes a major component of cardiovascular morbidity and mortality. A recent study by Akar et al. (2005) implicated left ventricular mitochondrial dysfunction in the generation of post ischemic arrhythmias. The relationship between susceptibility to develop atrial arrhythmias and mitochondrial function in atrial myocytes was investigated by Ad et al. (2005). It was shown that decreased MTT-reduction in conditions of simulated ischemia, similar to those applied in the present study, are associated with high susceptibility to develop postoperative atrial fibrillation following coronary bypass grafting operations in patients.

MTT has been widely used in the assessment of cardiac myocyte injuries, and the effect of cardiotoxic agents, both in cultured myocytes (Gomez et al., 1997; Green and Leeuwenburgh, 2002; Mojzisova et al., 2006) and ex vivo (Zheng and Hu, 2005). Susceptibility to ischemia has been studied using MTT in the context of the effect of preconditioning (Ghosh et al., 2000; Loubani and Galinanes, 2002). The assessment of the metabolic status by MTT has been further developed by Ad et al. (2005).

In the latter study, performed on human atrial biopsies, MTT-reduction in each biopsy was assessed in conditions of oxygenation versus simulated ischemia. The authors showed that the ratio between MTT-reduction in simulated ischemia and oxygenated conditions was associated with the susceptibility to develop atrial fibrillation. This result implied that this ratio reflects the status of the cellular metabolic reserves. In the present study, we took a similar approach to assess the cardiac effect of CEM.

It is now necessary to examine whether the ex vivo impairment of the ischemic response shown in the present study is associated with increased in vivo susceptibility to major ischemic events, and how exposure to low-dose CEM interacts with other cardiac injuries and heart diseases.

In summary, we found that an environmental agent, CEM, administered at a low dose, causes occult toxicity in the rodent heart. This toxicity is not expressed as long as the heart tissue receives adequate supplies of glucose and oxygen. When the tissue is challenged by an ischemic stimulus, the toxic effect surfaces, and the tissue shows increased susceptibility to ischemia, reflected by a decrease in MTT reduction to formazan. Such occult toxicity should be of concern to the environmental toxicologist. It is conceivable that various environmental stimuli do not exert an overt toxic effect on the examined tissues, but impair their ability to cope with common challenges, such as cardiac ischemia.