Bis(2-chloroethoxy)methane-Induced Mitochondrial and Myofibrillar Damage: Short-Term Time-Course Study

June Dunnick*,1, JoAnne Johnson{dagger}, John Horton{dagger} and Abraham Nyska{dagger}

* Environmental Toxicology Program, and {dagger} Laboratory of Experimental Pathology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

Received April 15, 2004; accepted June 6, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cardiotoxicity induced by 2-, 3-, 5-, and 12-day dermal administration of 400 and 600 mg/kg/day of bis(2-chloroethoxy)methane to F344/N male and female rats was characterized. The severity and incidence of lesions were similar among males and females and in all three regions of the heart examined (atrium, ventricle, interventricular septum). Damage induced by bis(2-chloroethoxy)methane consisted of time-related development of myofiber vacuolation, necrosis, mononuclear-cell infiltration, fibrosis, and atrial thrombosis. Changes were pronounced at day 2, increased in severity at day 3, appeared to decrease at day 5, and resolved by study-day 16 that corresponded to 12 dosings. Ultrastructural analysis of 2- and 5-day 600 mg/kg/day-treated females elucidated the primary site of damage, the mitochondrion, and two types of vacuolation, one that formed as damaged mitochondria became devoid of cristae and their bounding double membranes became reduced to singleness, and the other manifested as distention of the sarcoplasmic reticulum. After the initial damage induced by bis(2-chloroethoxy)methane, or its metabolite, thiodiglycolic acid, protective mechanisms within the heart were apparently initiated, enabling it to cope with the continued exposure to the toxicant while eliminating some damaged myofibers.

Key Words: bis(2-chloroethoxy)methane (CEM); thiodiglycolic acid; cardiotoxicity; vacuolation; mitochondria; sarcoplasmic reticulum (SR); recovery.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bis(2-chloroethoxy)methane (CEM), an organic compound from which polysulfide polymers used in sealant applications are generated, causes heart toxicity in rodents, characterized by cytoplasmic vacuolation of myocytes, necrosis, and inflammation (Dunnick et al., 2004Go). Investigators have hypothesized that thiodiglycolic acid, a metabolite of CEM, may be the compound that induces cardiotoxicity by damaging mitochondria and inhibiting their function (NIEHS Contract NO1-ES-75407, 2002Go; Visarius et al., 1998Go).

Other chemicals metabolized to thiodiglycolic acid that also produce heart toxicity in rodents and/or humans include the drug Ifosfamide (Visarius et al., 1998Go), monochloroacetic acid (National Toxicology Program, 1992Go), chloroacetaldehyde (Joqueviel et al., 1997Go), trichloroethane (Yllner, 1971Go), trichloroethylene (Anderson et al., 1987Go), 1,1-dichloroethylene (Anderson et al., 1987Go), cyclophosphamide (Joqueviel et al., 1997Go), vinylidene chloride (Jones and Hathway, 1978Go), and vinyl chloride (Green and Hathway, 1975Go). Since thiodiglycolic acid is a metabolic product of several chemicals in humans and/or rodents (Hofmann et al., 1991Go), our murine model of CEM-induced cardiotoxicity may also serve as an investigative tool for other chemical-induced heart damage.

In this article we describe the short-term sequential histological and ultrastructural alterations in the heart of F334/N rats exposed to CEM for 2, 3, 5, and 12 days and suggest that the mitochondria constitute the primary subcellular target for this chemical. The rat was selected as a model to characterize this heart toxicity, because a previous study showing that it is more responsive than the mouse to CEM-induced heart toxicity (Dunnick et al., 2004Go) is consistent with the finding that rats produce more thiodiglycolic acid than mice (Jones and Hathway, 1978Go; NIEHS Contract NO1-ES-75407, 2002Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemical and dose formulation. Bis(2-chloroethoxy)methane (CAS No. 11-91-1; lot B004160277) (Karl Industries, Aurora, OH) was found to be 98.5% pure (National Toxicology Program, 2000Go). Solutions of CEM were prepared in 95% ethanol for dosing by dermal administration daily, excluding weekends, for two weeks plus two consecutive dosages before the last sacrifice on study-day 16, at which animals had received a total of 12 doses. Fur from the site of application was clipped weekly. Stock solutions prepared at concentrations of 0, 800, and 1200 mg/ml were stored in amber glass bottles at room temperature. The administrations were applied to the skin of the rats at 0.5 ml/kg body to deliver doses of 0, 400, or 600 mg/kg body weight. All dose formulations were determined to be within ±10% of target concentrations. Approximately 45% of a dermal dose of CEM is adsorbed (NIEHS Contract NO1-ES-75407, 2002Go).

Animals and experimental design. Male and female F344/N rats (Taconic Laboratories, Germantown, NY) were placed on study at five weeks of age and housed one per cage in polycarbonate cages in rooms maintained at temperatures between 69 and 75°F with 35–65% relative humidity and a 12-h light/dark cycle. Control and treated groups received irradiated NTP-2000 diet (Zeigler Brothers, Gardners, PA) ad libitum. Body weights were recorded weekly. Groups consisted of 10 rats/sex/dose for sacrifice on study day 2, 3, 5, and 16 for histopathological assessments. An additional three animals per dose per sex were added for days 2 and 5 for collection of heart for electron microscopy. Sacrifice was approximately 2–3 h after the morning dosing. Left atria, left ventricles, and interventricular septa were collected from all rats. Animal husbandry and handling were conducted in accordance with NIH guidelines (Grossblatt, 1996Go).

Pathology and clinical chemistry. Core-study animals, those used for histopathology, were euthanized with carbon dioxide at the completion of dosing on day 2, 3, 5, and 16. Blood from rats anesthetized with a carbon dioxide/oxygen mixture was collected on study-day 5 for clinical pathological determinations of creatinine kinase (CK) and troponin T. Blood was drawn from the retro-orbital sinus, collected into serum separator tubes, and centrifuged, and sera were harvested for CK and troponin T analyses. The troponin T assessment uses an electrochemiluminescence immunoassay technology of the Roche Elecsys 2010 immunoassay analyzer.

At necropsy, all organs and tissues were examined for grossly visible lesions. For light microscopic examination, the heart was fixed in 10% neutral buffered formalin, processed, trimmed, embedded in paraffin, sectioned to a thickness of 4–6 µm, and stained with hematoxylin and eosin (H&E). A semiquantitative grading scheme was used to evaluate the extent of the lesions in the heart sections as follows: minimal (grade 1), lesions involved less than 10% of the heart section; mild (grade 2), 11–40%; moderate (grade 3), 41–80%; marked (grade 4), 81–100%.

Masson's Trichrome stain was applied to demonstrate myocardial necrosis and fibrosis (Luna, 1968Go). ApopTag staining, chosen to identify apoptosis, revealed normal nuclei as blue and apoptotic nuclei as brown (Herman et al., 1997Go; Zhang et al., 1996Go, 1999Go). In the ApopTag staining method, the terminal deoxyribonucleotidyl transferase nick end-labeling (TUNEL) assay is utilized.

Immunohistochemistry. An immunohistochemical stain was used to identify troponin T (cTnT) in heart muscle as previously described (Dunnick et al., 2004Go). The loss or partial loss of cTnT in the myofibers, based on the percentage of myocardial cells exhibiting composite cTnT loss within the entire heart section, was semiquantitatively scored using the following scale: no cells exhibiting cTnT loss = 0 (0), 1–25% cells = 1st quartile (1), 26–50% cells = 2nd quartile (2), 51–75% cells = 3rd quartile (3), and 76–100% cells = 4th quartile (4) (Dunnick et al., 2004Go; Herman et al., 1999Go).

Electron microscopy. Since light microscopy revealed damage throughout the heart of females and males, only two regions, left ventricle (LV) and interventricular septum (IS), were analyzed from 2-day and 5-day CEM-high-dose (600 mg/kg)-treated females and their corresponding controls. Following swift excision, the tissues were immersed immediately in fixative (3% glutaraldehyde [Ladd Research, Burlington, VT] buffered in 0.1 M sodium cacodylate [Electron Microscopy Sciences, Fort Washington, PA], pH 7.2), cut into 1-mm cubes. Following 2–3 days storage in the fixative, specimens were rinsed in buffer, postfixed in cacodylate-buffered 1% osmium tetroxide (Electron Microscopy Sciences), en bloc-stained in 2% aqueous uranyl acetate (Ted Pella, Inc., Redding, CA), dehydrated through a series of graded alcohols and propylene oxide, and embedded in Polybed 812 (Polysciences, Warrington, PA). At least three blocks from each region of each animal were analyzed. Semithin (1/2 µ) sections stained with 1% toluidine blue + 1% sodium borate were scanned light microscopically to locate regions containing longitudinal fibers and vacuolations. Ultrathin (90 nm) sections were cut from these regions, placed on 150-mesh copper grids, stained with 5% uranyl acetate followed by Reynold's lead citrate, and examined in a Philips EM 400 electron microscope.

Statistical analysis. Clinical chemistry and hematology data, which typically have skewed distributions, were analyzed using nonparametric multiple comparison methods of Shirley (1977)Go and Dunn (1964)Go.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Body Weights, Mortality, Clinical Signs
Mean body weights of treated rats were not significantly different from those of controls. All animals lived until the termination of the study. No treatment-related clinical signs of toxicity were observed.

Clinical Chemistry and Pathology
No differences in serum CK or cTnT levels were detected between control and treated animals (data not shown).

Histopathologically, at all time points examined, CEM was found to induce heart damage that changed in nature according to the day of sacrifice (Table 1). Treatment-related histopathological findings included vacuolation, myocardial necrosis, mononuclear-cell infiltration, fibrosis, and atrial thrombosis. The morphological characteristics were described previously (Dunnick et al., 2004Go). There were no other treatment-related lesions.


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TABLE 1 Histopathological Findings in the Hearts of F344 Rats Exposed to Bis(2-chlorethoxy)methane Administered by Dermal Exposure in 95% Ethanol Daily for 2, 3, 5, and 12 Days

 
The histopathological findings are presented in Figure 1. Myocardial necrosis was diagnosed when the sarcoplasm of the cardiomyocytes was homogenous and/or shrunken, with small and hyperbasophilic myocytic nuclei sometimes present. As seen on H&E slides, the altered staining pattern indicative of myocytic necrosis consisted of uniform hypereosinophilia and the absence of banding (Fig. 1E). On trichrome preparations, necrotic myofibers appeared dark red (Figs. 1H and 1L), while, with troponin-T (cTnT) immunostaining, the necrotic myofibers lost the normally present brownish staining (Figs. 1F, 1J, and 1N). The myofiber necrosis was associated with cytoplasmic vacuolation (Figs. 1E, 1I, and 1M), and mononuclear-cell infiltration was sporadically TUNEL-positive (Figs. 1K and 1O). In the five-day samples, the presence of collagen deposition was indicated by the bluish Masson's trichrome-stained fibrils (Fig. 1P).



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FIGS. 1A-D. Light microscopy of time-related progression of cardiotoxicity. Female F344 rats: controls (Figs. 1–4) and those administered two (Figs. 5–8), three (Figs. 9–12), and five (Figs. 13–16) daily dermal doses of 600 mg/kg/day bis(2-chloroethoxy)methane. Interventricular septum. x200.

FIGS. 1A, E, I, M. Hematoxylin and eosin. In the two-day photos, the myofiber necrosis is relatively prominent (arrows) with an inflammatory reaction and scant vacuolation. In the three- and five-day photos, myofiber vacuolation and mononuclear infiltration are prominent (arrows).

FIGS. 1B, F, J, N. Troponin-T (cTnT) immunostaining. In the two-, three-, and five-day photos, the areas of myofiber necrosis, vacuolation, and inflammatory infiltration are identified by loss of cTnT staining.

FIGS. 1C, G, K, O. TUNEL immunostaining. In the three- and five-day photos, sporadic mononuclear cells exhibit TUNEL-positive nuclei (arrows).

FIGS. 1D, H, L, P. Histochemical Masson's trichrome staining. In the two- and three-day photos, necrotic myofibers appear homogeneously red (arrows). In the five-day photos, collagen deposition appears blue (arrows.).

 
Control and treated rats also exhibited sporadic foci of mononuclear-cell infiltration, which is consistent with spontaneous cardiomyopathy, a common age-related change (Ruben et al., 2000Go).

Day-by-Day Distribution of Lesions
The severity and incidence of CEM-induced heart lesions was similar between male and female rats (Table 1) and among atrial, ventricular, and interventricular-septal regions. After two days of dosing, a minimal to mild cytoplasmic vacuolation and myocardial necrosis could be seen in the hearts of male and female rats treated with 400 and 600 mg/kg/day. The severity of mononuclear-cell infiltration was increased in the treated groups compared to the background incidence observed in controls.

At three days of dosing, the severity of the treatment-related cytoplasmic vacuolation, myocardial necrosis, and cellular infiltration had increased. Both the incidence and average severity grades of cytoplasmic vacuolation, myocardial necrosis, and cellular infiltration were higher in day 3 than day 2 600 mg/kg/day-treated males and females. In addition, fibrosis and thrombosis were seen in some of the 600 mg/kg treated rats.

At day 5, there was a reduction in the CEM related heart lesions including a reduction in the incidence and/or severity of the cytoplasmic vacuolation, mononuclear cell infiltration, and necrosis compared to that observed in day 3 male and female hearts. However, all males and females at 600 mg/kg and about half of the animals at 400 mg/kg exhibited fibrosis in the hearts at day 5.

By day 16, the treatment-related cytoplasmic vacuolation and necrosis observed on days 2, 3, and 5 had resolved. The severity of the mononuclear-cell infiltration in the treated rats had now returned to the baseline level and was similar to that observed in the control rats. There was no evidence for treatment-related thrombosis at day 16. A decrease in the incidence and severity of fibrosis observed in the heart of treated male and female rats was also apparent.

Electron Microscopic Characterization of Lesions
In the left ventricle (LV) and interventricular spetum (IS) regions of 2-day and 5-day controls, relative normality of mitochondrial arrays and ultrastructure of organelles was observed. The mitochondria were aligned characteristically in an elongate pattern alongside intact myofibrils showing matched banding and contained well-defined inner and outer bounding membranes; numerous, mainly intact cristae; and matrices (Fig. 2A) that were ordinarily electrondense.



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FIG. 2. Transmission electron micrographs. (A) Mitochondrial alignment and ultrastructure and myofibrillary banding appear normal in five-day F344 female control; left ventricle. (B) Mitochondria from five-day 600 mg/kg/day-treated F344 female appear swollen and damaged with breakage and deterioration of cristae resulting in electronlucency. Some rounded clumping of mitochondria (arrows) and offsetting of dark Z-band alignment (arrowheads) appear to be occurring; inverventricular septum. Bars = 3 µ. M = mitochondrion; My = myofiber.

 
Observations of cardiac muscle from rats exposed to high-dose CEM (600 mg/kg/day) revealed, however, that after two days of treatment, in both the LV and IS regions, damage to most, but not all, myocytes had occurred. Both regions examined appeared equally adversely affected by the chemical insult.

Alterations in mitochondria constituted the most prominent feature of this lesion (Fig. 2B), but distention of sarcoplasmic reticulum (SR) (Figs. 4 and 5), myofibrillary degeneration (Fig. 4), and occasional Z-banding misalignments (Fig. 2B) were also apparent. Swelling and a looser arrangement of the endothelium were indicative of inflammation, and mononuclear-cell infiltration was pronounced. A few necrotic, disintegrating myocytes contributed to membranous debris in the interstitial spaces. Severe disruption of mitochondrial ultrastructure culminating in disintegration and vacuolation (Fig. 3) was the most conspicuous damage that occurred in affected myocytes in 2-day and 5-day 600 mg/kg/day-treated animals. Abnormalities comprised occasional rounded clusterings of mitochondria no longer arranged in their characteristic pattern of linear alignment between the myofibrillary bundles (compare Fig. 2A with Fig. 2B), fragmented cristae, mitochondrial electronlucency with minimal amorphous electrondense matter resulting from loss of damaged cristae, inclusions consisting of concentric or nonconcentric whorls and/or other unusual membranous profiles, swelling of mitochondria, the appearance of widely scattered megamitochondria (Fig. 4B) (Cheville, 1994Go; Matsuhashi et al., 1998Go), and vacuolation.



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FIG. 4. Transmission electron micrographs. (A) Swollen mitochondrion containing fragmented cristae shows separation of double bounding membrane on one side (arrow). Note also the myofibrillary (My) degeneration at this site. F344 female, two-day, 600 mg/kg/day, interventricular septum. (B) Megamitochondrion seen adjacent relatively normal-sized mitochondrion. F344 female, two-day, 600 mg/kg/day, left ventricle. Bars = 1 µ.

 


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FIG. 5. Transmission electron micrograph. Vacuolation of sarcoplasmic reticulum (V) appears contiguous with dilation of the nuclear envelope (arrow). F344 female, two-day, 600 mg/kg/day, left ventricle. Bar = 1 µ.

 


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FIG. 3. Transmission electron micrograph. Vacuolation of several mitochondria with some bounding double membranes intact but others reduced to singleness (e.g., inset of profile below center to left; single arrow denotes singleness of bounding membrane; the two arrows indicate doubleness; bar = 0.5 µ). Note that many of the vacuolating structures show only wispy profiles of remaining, disintegrating cristae; some amorphous, electrondense material; and electronlucent areas completely devoid of ultrastructure. *Indicates mitochondrial profiles in close apposition with electronlucent space between, possibly coalescing. F344 female, five-day, 600 mg/kg/day, interventricular septum. Bar = 1 µ.

 
As damage progressed and disintegration of the mitochondria began to occur, cristae disappeared, and the typical double bounding membrane deteriorated and separated (Fig. 4A), leaving, in many, only a single bounding membrane or a single bounding membrane on one side and double membrane on another (Fig. 3, inset). Coalescence of the resulting vacuolating or vacuolated profiles may have been occurring (Fig. 3) leading to the larger vacuolated spaces seen in better overview at the light microscopic level.

After five days of treatment, the damage at the electron microscopic level appeared approximately the same as that seen in day 2 females, although megamitochondria were much more scarcely seen, and more phagocytic removal of myocytic debris could be observed.

The rare megamitochondria, also termed macromitochondria (Cheville, 1994Go) or giant mitochondria (Mashimo et al., 2003Go) appeared in both the ventricular and interventricular septal regions, predominantly in the two-day treated animals (Fig. 4B). These unusual mitochondria have been described as having a cross-sectional area larger than five times that of normal ones (Mashimo et al., 2003Go). Even considering the swelling apparent in nearly all damaged mitochondria (~ two times the size of control mitochondria), we estimated visually that the megamitocohondria were enormous, 5–10 times the size of those not so grossly swollen. Their matrices were strikingly electronlucent as the cristae were greatly reduced in number; some scattered, electrondense, amorphous material could be seen between remaining cristae.

While some cristal alterations and slight swelling of mitochondria occurred rarely to occasionally in controls, this artifactual background damage was estimated visually, on a scale of 1–4, to be of a minimal degree, 1, while degrees of damage of 3–4 were most often seen in the two-day and five-day treated animals. No megamitochondria, vacuolation, or unusual phagocytic activity were observed in controls.

Distention of the SR producing a second type of vacuolation was prominent in several areas in both two-day and five-day treated females (Fig. 5). Distinguishing between vacuolation of the single membrane–bounded SR and vacuolation of mitochondria damaged to the extent of retention of only one bounding membrane was occasionally difficult and could be performed with confidence only by some visible linkage of the SR network to the nuclear envelope or by observing the SR in its typical position near the myofibrils. Vacuolation of the SR occurred most frequently in contiguity with a swollen nuclear envelope (Fig. 5); that mitochondria in nearby areas did not usually appear severely damaged to the point of dissolution of their bounding membranes lent further credence to the identification of this type of vacuolation as sarcoplasmic reticular.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The various techniques employed in this study allowed elucidation of the time-course of the heart damage induced by CEM, the subcellular sites of deterioration, and the initiation of damage reduction that may have resulted from some type of protective response to the chemical insult (Table 2). Bis(2-chloroethoxy)methane induced observable cardiotoxicity in the F344/N rat after only two days of dosing, but by study day 16 a resolvement of the manifestations of the lesion had occurred. Staining by H&E allowed characterization of the damage as cytoplasmic vacuolation, necrosis, and mononuclear-cell infiltration. When the lesions peaked in severity after three days of dosing, a fibrotic reaction occurred in response to the tissue damage. By day five, the fibrotic response was more pronounced, but the initial damage of cytoplasmic vacuolation was becoming less severe, indicating that no new treatment-related heart damage was occurring. By study day 16, light microscopic evidence for active myofiber alteration, such as vacuolation and necrosis, was no longer apparent.


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TABLE 2 Summary of BCEM-Induced Cardiac Damage in 600 mg/kg Group: Time-Course Study of Damage and Defensive Response

 
Troponin staining indicated that the integrity of the myofibers was lost, but the lack of measurable levels of serum troponin may suggest that the levels in blood were too low or the analysis after five days was too late, so that the optimal diagnostic window reflecting the greatest magnitude and duration of cardiac damage was missed (Wallace et al., 2004Go). The lack of TUNEL staining of degenerating myocardial cells suggests that the cardiac damage, at least at the stage of evaluation, was not associated with prominent myofiber death.

To characterize the morphological manifestations of the heart toxicity beyond the level possible with conventional histological methods, an electron microscopical examination was performed. This analysis of left ventricle and interventricular septum revealed vacuolations arising predominantly from disintegrating mitochondria and, to a lesser extent, expanding SR. Mitochondrial disintegration and vacuolation apparently led to subsequent myofibrillary degeneration and destruction and eventual myocytic rupture. Swelling of the SR, constituting the less prevalent type of vacuolation, was considered a degenerative change, known to be one of the signs of cellular injury due to loss of membrane function in maintaining water balance (Cheville, 1994Go). Since we did not observe proliferation, or reduplication of the SR membrane, typified by bundled tubules (Cheville, 1994Go) and a hallmark of an adaptive response, we inferred that the CEM-induced damage was indeed degenerative. That the distention of the SR was found most often contiguous with a swollen nuclear envelope may indicate some type of alteration of protein import or export, but whether the blebbing of the nuclear envelope itself represents a pathological response is debatable (Ghadially, 1997Go). We believe that it may have been induced by treatment, since distensions of the nuclear membrane were most often seen contiguous with swollen SR membrane. In this study, CEM caused such damage in the heart mitochondria of the F344 rat as to be conspicuous after only two days of chemical exposure, but never to such a degree that the animals showed clinical signs of cardiotoxicity, such as weight loss, or expired as a result of the treatment. Mitochondrial impairment may have been initiated even before the morphological manifestations were detectable. Damage to mitochondria can impair energy production, release triggers for apoptosis such as cytochrome c, and/or decrease the ability to eliminate oxygen radicals through the manganese-containing mitochondrial superoxide dismutase. Loss of mitochondrial function may also result in an accumulation of toxic compounds in the myocyte, such as those produced from acidosis or a buildup of lactate (Lesnefsky et al., 2001Go). Generally, an excess of mitochrodrial function is believed to exist in the heart so that it is able to compensate for relatively minor damage. A decrease in mitochondrial function of at least 30–50% is ordinarily required before lower rates of energy production occur (Lesnefsky et al., 2001Go). Most likely this degree of damage was not attained in our study, since the animals retained a sufficient reserve of energy-generational capacity to survive asymptomatically and without any sign of apoptosis.

In a previous study, we reported that CEM induced heart toxicity characterized by cytoplasmic vacuolation, necrosis, and inflammation, in the heart in F344 rats after dosing with 400 or 600 mg CEM/kg for up to 90 days (Dunnick et al., 2004Go), All male and female rats at 600 mg/kg and some of the females at 400 mg/kg died before the end of that study. In the present 16-day CEM time course study, females and males appeared equally affected in all three regions of the heart, and by day 16, the lesion had resolved. We hypothesize, therefore, that after the initial damage inflicted by CEM that occurs during the first few days of chemical exposure, the animal launches protective mechanisms to prevent further myocardial damage. Sometime between days 16 and 90, however, the protective defenses become somehow unable to ward off the insult.

The defensive mechanism(s) induced in the rat heart after the chemical exposure could be activated or mediated by increases in levels of superoxidase dismutase for protection from injury caused by generation of free radicals from CEM and/or its metabolites, such as thiodiglycolic acid. This process has been proposed as a possible protective response involving other chemicals, such as doxorubicin (Childs et al., 2002Go). Combined in vivo and in vitro studies, using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical-induced cardiac injury, demonstrated that the ex vivo administration of the compound ramipril induced delayed cardioprotection, possibly related to induced synthesis of proteins, such as heat-shock protein and antioxidants (Jin and Chen, 1998Go). Investigations using the model of alcohol-induced cardiotoxicity in rat suggested that the mitochondria of the heart, in contrast to those of the liver or brain, exhibit significant increases in mitochondrial enzymatic activities. Increases were noted in levels of citrate synthase; complexes I, III, IV, and V; and mitochondrial DNA, which are indicative of an increase in mitochondrial number, considered to be an adaptive response (Marin-Garcia et al., 1995Go). In vitro studies with different doses of ethanol indicated that only high doses of 200 Mm induced formation of giant or megamitochondria (Mashimo et al., 2003Go). This mitochondrial hypertrophy is considered to be an adaptive response to switch energy-generating myocardial metabolism to some specialized function.

While cardiac failure arises from many general, diverse causes (Jessup and Brozena, 2003Go), mitochondrial damage and malfunction are widely associated with human heart disease, including dilated and hypertrophic cardiomyopathy, ischemic and alcoholic cardiomyopathy, and other syndromes (Marin-Garcia et al., 1995Go). Mitochondrial defects may arise from mutations or deletions in the mitochondrial genome (Marin-Garcia and Goldenthal, 2002Go) or through free-radical damage suggested to be the mechanism of action of doxorubicin (Childs et al., 2002Go; Cummings et al., 1992Go). Megamitochondria have been closely associated with the generation of free radicals (Wakabayashi et al., 1997Go).

Megamitochondria have been observed in chemical toxicity and may arise by fusion of adjacent mitochondria or suppression of division leading to extreme swelling (Cheville, 1994Go; Matsuhashi et al., 1998Go; Sudarikova et al., 1997Go). Our observations of megamitochondria chiefly in the two-day animals may reflect a greater degree of pathological response to the toxicity at this time point, consistent with the light microscopic findings, which were showing some reduction in parameters of damage by day 5. Other lesion characteristics were similar, however, between the two-day and five-day high-dose treated animals. Since electron microscopy allows visualization of but a tiny fraction of the area revealed by light microscopy, the sampling size, while large by ultrastructural standards, was probably insufficient to give a complete overview.

Impairment of the functioning of the mitochondrion, the principal site for the metabolism of carbohydrates and fats, could lead to heart toxicity and cellular death through the loss of metabolic capacity or the release of substances that are toxic or trigger the apoptotic pathway (Lesnefsky et al., 2001Go). Further investigation is required to determine exactly how CEM or its metabolite(s), such as thiodyglycolic acid, actually effectuates the observed mitochondrial damage. One study suggests that thiodiglycolic acid can interfere with oxidation of palmitic acid, a long-chain fatty acid, but not the metabolism of succinic acid that would be metabolized by the Kreb's cycle (Visarius et al., 1998Go). The dicarboxylic acid structure of thiodiglycolic acid may enable it to compete with the fatty-acid metabolic pathways that involve activation of fatty acids by long-chain fatty acid CoA synthetase, followed by formation of acylcarnitines by carnitine palmitoyltransferases (Lesnefsky et al., 2001Go). This proposed mechanism might in part explain the CEM-induced heart toxicity and that of other chemicals metabolized to thiodiglycolic acid. Even if CEM disrupts energy generation in the heart, however, this interference does not appear to explain completely the mitochondrial damage observed, because the pathway for the Kreb's cycle would still be intact and capable of generating energy.

Based on this investigation and the previous 90-day study (Dunnick et al., 2004Go), we suggest that CEM may cause a biphasic heart toxicity in F344 rats. After dosing for 2–3 days, widespread evidence of cardiotoxicity was apparent, including myofiber disintegration, vacuolation, necrosis, and cellular infiltration. By 16 days, the areas of vacuolation were no longer present because they were replaced by fibrosis; or the heart developed resistance by generating protective, possibly antioxidant, mechanisms; and/or the heart regenerated myocytes (Urbanek et al., 2003Go). The observation of heart damage at two, three, and five days of dosing, not at study-day 16, but again at 90 days, may be explicable by the initiation within the heart of some kind of temporary defensive adaptive response to the toxic chemical insult.


    ACKNOWLEDGMENTS
 
We gratefully recognize Dr. J. Ryan and Dr. M. Hejtmancik of Battelle-Columbus Operations for their excellent supervision of the in-life phase of the studies; and Dr. Connie Cummings of Pathology Associates International and Dr. James Nold of Glaxo-Smith-Kline for consultation in interpretation of electron micrographs, and Dr. Robert Sills and Dr. Hiroshi Satoh, National Institute of Environmental Health Sciences, for their review of the manuscript.


    NOTES
 

1 To whom correspondence should be addressed at NIEHS, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709. Fax: (919) 541-4255. E-mail: dunnickj{at}niehs.nih.gov.


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 DISCUSSION
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