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Experimental Therapeutics |
Department of Biology, Beckman Research Institute at the City of Hope [G. L. F., B. G., J. M.] and Department of Pediatrics, City of Hope [X. L.] Duarte, CA 91010; and Northwestern University, Evanston, Illinois 60201 [W. T.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous reports on in vitro studies have implicated the C-13 hydroxy metabolite, dxol, as a major component in the development of cardiotoxicity (4 , 5) . These data contrast with tumor cell killing in which the C-13 hydroxy metabolite is less toxic against tumor cells examined in tissue culture systems (6, 7, 8, 9) . Several ubiquitous enzymes such as CBR(s) and aldehyde reductase(s) metabolize dox to dxol, implicating these enzymes in the development of cardiotoxicity (10 , 11) . HCBR is a cytosolic enzyme that reduces dox to dxol in the presence of the cofactor, NADPH (11 , 12) . In this report, we developed a transgenic mouse model using a mouse heart-specific promoter (13) to express HCBR in the hearts of transgenic mice and measured morphological and ECG changes during the development of acute and chronic dox-induced cardiotoxicity.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MHC, was kindly
supplied by Dr. Jeffrey Robbins (Children’s Hospital Medical Center,
Cincinnati, OH; Ref. 13
). The coding region of the HCBR
cDNA was amplified from the plasmid pHCR 311 (14)
using
the PCR and synthetic primers containing a SalI linker at
the 5' ends. The modified HCBR cDNA was cloned into the SalI
site of pMHC. DNA sequencing on an ABI Model 373 fluorescent DNA
sequencer verified the sequence and orientation. The expression vector
containing the HCBR cDNA was digested with NotI to linearize
the DNA. The fragment containing the heart-specific promoter and HCBR
cDNA was purified on a 10–40% linear sucrose gradient, was
concentrated, and was stored in TE buffer [10 mM
Tris (pH 7.50) and 0.1 mM EDTA] at -20°C. The
final concentration was adjusted to 2.5 µg/ml.
Transgenic Mice.
All of the animal procedures were approved by the American Association
of Accreditation of Laboratory Animal Care and were certified by an
institutional animal care committee. B6C3F1 mice were obtained from
Taconic (Germantown, N.Y.) and were used to produce the transgenic
lines. Standard procedures were used to produce transgenic mice in the
transgenic core facility at the City of Hope (Duarte, CA; Ref.
15
). Positive HCBR founder transgenic mice were identified
by in-gel Southern analyses on genomic DNA isolated from 0.3-cm
tail biopsy and/or by PCR analyses (16)
using primers to
the MHC promoter region and to the HCBR coding sequence. The HCBR
cDNA was used as a probe for Southern and Northern analyses.
Enzyme Assays.
Ten to 20 mg of tissue were homogenized in 1 ml of 10 mM
Tris-HCl (pH 7.4) and 1 mM EDTA and were centrifuged at
16,000 x g for 10 min at 4°C. The
supernatant was used for enzyme assays. Protein concentration was
determined using a Protein Assay Kit (Bio-Rad, Hercules, CA) using BSA
as the protein standard.
Creatine Kinase Activity.
Animals were sedated using approved veterinary procedures. The thoracic
cavity was opened and blood was drawn from the left ventricle. Blood
samples were kept on ice until all of the samples were collected, then
were centrifuged at 16,000 x g for 5 min at
4°C. Serum was collected and stored at -20°C until assayed.
Creatine kinase was determined using a CK 10 Kit (Sigma, St. Louis,
MO).
CBR Activity.
CBR activity was determined from tissue extracts by measuring the
reduction of cytochrome c at 550 nm at 25°C using a Uvikon
930 spectrophotometer (Kontron Instruments/Research Instruments
International, San Diego, CA; Ref. 17
). Extract-containing
enzyme was added to buffer consisting of 0.1 M
potassium phosphate (pH 6), 0.2 mM menadione, 1.5
mM cytochrome c, and 0.2
mM NADPH. The CBR inhibitor, rutin (20
µM), was added to the reaction mixture 5 min
prior to starting the reaction with NADPH. The amount of enzyme
activity attributed to CRB is reported as rutin-inhibitable activity.
dxol Formation.
Dxol was measured by incubating cell extracts (10–100 µg) at 37°C
in 0.1 M potassium phosphate (pH 6), 0.65 mM
dox, and 0.5 mM NADPH (17
, 18)
. The reaction
was stopped and extracted with 0.6 ml of chloroform:isopropanol (1:1).
After centrifugation, 0.5 ml of the organic layer was transferred to a
new tube, and the sample was dried in a Speed-Vac concentrator. The
dried sample was dissolved in HPLC mobile phase buffer and
analyzed by HPLC using a phenyl column (19)
.
EM.
Mice, 8–12 weeks old, were injected i.p. once with 15 mg/kg dox
(Sigma) for acute dox treatment and i.p. with 5 mg/kg/wk for chronic
dox treatment. After 96 h (acute treatment) or 4 weeks (chronic
treatment) the animals were anesthetized, the hearts were perfused with
NaCl solution (0.8%) and then fixative (4% paraformaldehyde/2%
gluteraldehyde in 0.1 M cacodylate buffer) via the right
atrium. Portions of the left ventricle were cut into
1-mm2 cubes and were washed three times in
cacodylate buffer. Samples were post-fixed at 4°C for 1 h in 1%
OsO4, washed 3 x in buffer,
dehydrated in alcohols, and embedded in Eponate. One-µm
sections were stained in 5% uranyl acetate, followed with Sato’s Lead
stain. The samples were viewed and photographed with a Philips
CM 10 transmission electron microscope. All of the samples were
coded and scored in a blind fashion. One representative picture was
taken for every 10–15 fields, and five pictures were taken for each
heart.
HCBR Polyclonal Antibody.
Polyclonal HCBR antibody was produced in rabbits using purified HCBR
protein isolated from Escherichia coli expressing a HCBR
cDNA.
Immunohistochemical Staining.
Fresh tissue was excised from mice, fixed in 10% buffered formalin
overnight, and embedded in paraffin (20)
. Polyclonal HCBR
antibody was used with dilutions of 1:2000–1:5000. The
antibody-antigen complex was detected by a modified ABC method (Vantana
Medical Systems Inc.) according to the manufacturer’s directions with
the chromagen, 3'3-diaminobenzidine.
ECG Telemetry.
ECGs were measured telemetrically in conscious, freely moving
mice by monitoring signals from surgically implanted transmitters
(model ETA-F20; Data Sciences, St. Paul, MN). Six implantable
telemetric biopotential transmitters, receivers, and analogue ECG
adapters were used to collect data into a MacLab charts program. Data
were collected three times a week using a MacLab/4 MKIII analogue
digital instrument and a Macintosh G3 series PowerBook computer.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Three transgenic founders (HCBR1, HCBR2, HCBR3) expressing
heart-specific HCBR were produced and were used to generate
heterozygote and homozygote transgenic lines. HCBR1 and HCBR2 heart
extracts displayed heart-specific menadione reductase activities of
2500 nmol/min/mg protein and 4700 nmol/min/mg protein, respectively,
corresponding to a 250- and 470-fold increase, respectively, over
nonexpresser controls (Fig. 1)
. A corresponding increase in HCBR mRNA was also observed in the HCBR
expressers (Fig. 1)
. Table 1
shows organ-specific CBR activity in HCBR3. There was a 700-fold
increase in heart-specific CBR activity in transgenic expresser line
HCBR3 and low activity in the other organs. Transgenic lines HCBR1 and
HCBR2 showed similar CBR activities in other organs (data not shown).
No significant increase in CBR activity was observed in any other
organs other than the heart. Heterozygotes with heart-specific HCBR
activity up to 7,000 nmol/min/mg appeared normal. Homozygotes were
produced with heart CBR activities from 5,000 to 10,000 nmol/min/mg.
Transgenics expressing HCBR >9,000 nmol/min/mg developed severe
cardiomyopathies. Hearts were enlarged, and the mice did not reproduce.
Heterozygotes with HCBR expression levels 
5,000 nmol/min/mg (HCBR2)
were used in experiments.
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shows immunohistochemical staining of HCBR in the hearts of
HCBR2 expressers. HCBR protein expression was myocyte-specific
and was expressed abundantly and uniformly in all of the myocytes.
Epithelial cells were not stained (Fig. 2A
,
arrows). The myocyte staining pattern was representative for
all of the HCBR expressers including the homozygotes. Fig. 2B
shows that the nonexpresser hearts stained negative for
HCBR protein.
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; Table 2
). In the heart, HCBR expressers converted 82% of dox to dxol within
0.5 h after injection compared with 5% conversion in the
nonexpressers (Fig. 3)
.
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shows the percentage of dxol in liver,
lung, spleen, and quadricep. A maximum of 22% dxol was observed in the
liver after 12–24 h. Kidney dxol percentages were also measured.
However, we were not able to obtain accurate measurements because of an
interfering peak. The metabolism of dox to dxol in the kidney appeared
similar to that in the liver. The percentage of dxol in other organs
was low and similar in the HCBR expressers and nonexpressers (Table 2)
.
Total dox + dxol Levels in the Hearts of
dox-treated Mice.
The amount of heart dox + dxol was determined for the mice
shown in Fig. 3
. Drug levels were averaged for hours 0.5–12, which
included 20 hearts each for the HCBR expressers and nonexpressers. The
HCBR expressers contained an average of 2124 ng/g heart tissue compared
with 517 ng/g heart tissue for the nonexpressers. These results show
that dxol concentrations rapidly increased in the HCBR expresser hearts
and were four times higher than in the nonexpressers over the 12-h time
period. Although the total drug concentration was four times higher in
the HCBR expressers over 12 h, it decreased 5-fold by 48 h to
a level similar to that of the nonexpressers.
Effect of Acute dox Treatment on Serum Creatine Kinase Levels.
HCBR expresser and nonexpresser mice (n = 5)
were given a single injection of dox (10 mg/kg, i.p.). Serum was
collected at 48 h and analyzed for serum creatine kinase activity.
HCBR expressers displayed a 60% higher level of serum creatine kinase
activity compared with the nonexpressers (Table 3)
. These data suggest that HCBR expressers are more sensitive to heart
or muscle damage than nonexpressers.
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. HCBR expressers displayed increased cardiac damage, which was five
times higher than the cardiac damage found in nonexpressers.
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. Fig. 5A
shows a dox-treated HCBR expresser exhibiting
extensive myofibril degeneration. A HCBR-untreated control is shown in
Fig. 5B
. Fig. 5C
shows increased vacuolization
with some myofibril degeneration in the dox-treated nonexpresser. An
untreated nonexpresser control is shown in Fig. 5D
.
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shows the survival data for the HCBR expressers and the nonexpressers.
The HCBR expressers died within 5 weeks, whereas the nonexpressers
survived for 12 weeks.
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shows representative ECG profiles collected each week
during chronic dox treatment of a HCBR expresser. The largest peak
represents depolarization of the ventricular myocardium resulting in
heart contraction. Immediately following the main peak is the T wave
(arrow), which represents repolarization of the heart. A broadening and
decreasing T wave correlates with the development of dox-induced
cardiotoxicity in mice (24
, 25)
. Fig. 7A
shows
that the T wave started to broaden by week 2 and was almost
undetectable by week 3. In addition to the change in the T wave, the
main peak dropped to 50% of its initial value. The main peaks for the
other two HCBR expressers dropped by 80%. These data indicate the
development of severe cardiomyopathy.
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Fig. 7B
shows ECG profiles in a nonexpresser mouse at 3-week
intervals. The data show that the T peak began to broaden by week 6–8
and continued to broaden with the development of cardiotoxicity. The
nonexpresser mice survived for 12 weeks, 2.5 times longer than the HCBR
expressers.
There were differences in the development of chronic cardiotoxicity between the HCBR expressers and nonexpressers. The main ECG peak in the control mice did not decrease as it did in the HCBR mice. The onset of cardiotoxicity was slower in the controls, and the mice showed a steady decline in their health as observed by their inactivity and coat texture. Necropsy examination showed enlarged hearts. There was not a build-up of fluid in the thoracic cavity before death as was observed in the HCBR expressers.
Mitochondrial Damage in Chronically Treated Mice.
HCBR expressers and nonexpressers (n = 4)
were treated chronically with dox (5 mg/kg, i.p.) for 4 weeks. Hearts
were processed for EM analysis as described earlier. The dox-treated
HCBR expressers displayed more extensive mitochondrial damage (Fig. 8A
, arrow) compared with the dox-treated
nonexpressers (Fig. 8B)
. These data were in contrast to the
data from acutely treated animals in which the major type of damage was
myofibril degeneration as shown in Fig. 5A
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dxol is the major metabolite of dox found in tissue (27) . It is formed by an enzymatic two-electron reduction of the C-13 keto group by cytoplasmic, NADPH-dependent aldo-keto reductases and short-chain dehydrogenases (10 , 17 , 21 , 22 , 28 , 29) .
Dxol seems to be more cardiotoxic than the parent drug. It inhibits many of the ion channel pumps. The calcium pump of the sarcoplasmic reticulum, the Na/Ca pump of the sarcolemma, and the proton pump of the mitochondria are inhibited by dxol but not by similar concentrations of dox (5 , 9) . There is also evidence that dox and dxol may act together at different sites to generate cardiotoxicity (30) . Lowering the level of dxol in vivo by inhibiting aldo-keto reductases with phenobarbital leads to less toxicity as measured by decreased creatine kinase levels (31) . Dxol also interacts with cis-aconitate and inhibits aconitase activity. This interaction causes the delocalization of iron from the active center of aconitase with reoxidation of dxol to dox (32) . This mechanism irreversibly inactivates aconitase and interferes with the aconitase-iron regulatory protein-1 function.
Dxol levels preferentially build up in the heart (30 , 33) , which suggests increased involvement of the C-13 hydroxy metabolite. Dxol build-up could be caused by lower transport through heart membranes, which is attributable to the more polar dxol molecule.
The HCBR transgenic mouse model allows one to exclude the potential membrane transport problems of using dxol, which may lead to lower drug concentrations in the heart (34) . Both acute and chronic dox schedules led to enhanced cardiotoxicity in the HCBR expressers; dxol levels increased rapidly and remained four times higher than the dox levels in the nonexpressers over 12 h. Total drug levels dropped 5-fold by 48 h and were similar in the expressers and the nonexpressers.
HCBR expressers showed five times more acute dox-induced cardiac damage as judged by EM along with a 60% increase in serum creatine kinase activity above that in nonexpressers. A single acute dose of dox caused heart damage 96 h after injection, which was mostly represented by myofibril degeneration. Chronic dox treatment (5 mg/kg/wk) showed mitochondrial swelling and degeneration after 4 weeks. The differences between acute and chronic cardiotoxicity could be attributable to different intercellular targets of dox and dxol or else to the higher concentrations of dxol. Transcriptional factors, heart-specific proteins, energy metabolism, and mitochondrial enzymes are sensitive to low concentrations of dox (35, 36, 37, 38) . Dxol levels in the hearts of the HCBR expressers were four times the levels of dox in the nonexpressers. The effect of this difference in metabolite concentration on mechanisms involved in cardiotoxicity is not known, but it appears to significantly advance the development of cardiotoxicity.
Dox-induced free radicals have been implicated in developing cardiotoxicity (39, 40, 41, 42, 43, 44, 45) . The C-13 hydroxy metabolite of dox can generate free radicals (46 , 47) . NADH dehydrogenase from heart mitochondria and heart sarcosomes containing NADPH cytochrome P450 reductase reduce dxol, generating superoxide anion. (47) . Dxol appears to produce 50% of the free radicals produced by dox (47) . The levels of dxol in the expresser hearts were four times the levels of dox in the nonexpresser hearts, providing a potential for a 2-fold increase in free radical formation. Enzymes that protect against the toxic effect of superoxide anion, such as the mitochondrial manganese superoxide dismutase, provide protection against dox-induced cardiotoxicity (48 , 49) .
Our data show more mitochondrial damage in the dox-treated HCBR expressers. Recent data suggest that several components of the mitochondrial electron transport chain, capable of generating superoxide anion, are involved in dox-induced cardiotoxicity. Dox-mediated inactivation of the NADH-dehydrogenase (complex I) in transgenic mice is protected by overexpression of manganese superoxide (50) . In yeast, cytochrome oxidase (complex IV), a source of superoxide anion, is necessary for dox-induced toxicity. Overexpression of mitochondrial manganese superoxide reduces the toxicity (48) .
In summary, we developed an in vivo transgenic mouse model system and showed that overexpression of HCBR in the heart leads to earlier development of dox-induced cardiotoxicity. The data show that the C-13 hydroxy metabolite of dox played a major role in cardiotoxicity and implicate CBR as a contributor to dox-induced human cardiotoxicity. The actual mechanisms involved were not determined but involve mitochondrial damage and leave open a role for direct drug metabolite interaction and free radical damage or combinations of both.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by Grant 9750100N from
the American Heart Association. DNA sequencing equipment was supported
by National Science Foundation Grant 9602945. 
2 To whom requests for reprints should be
addressed, at Department of Biology, Beckman Research Institute at the
City of Hope, 1450 East Duarte Road, Duarte, CA 91010.
3 The abbreviations used are: dox,
doxorubicin; dxol, doxorubicinol; HCBR, human CBR; CBR, carbonyl
reductase; MHC, myosin heavy chain; ECG,
electrocardiograph/electrocardiogram; EM, electron microscopy;
HPCL, high-performance liquid chromatography.
Received 1/26/00. Accepted 7/19/00.
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REFERENCES |
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T segment as the most reliable electrocardiogram parameter for the assessment of Adriamycin-induced cardiotoxicity in the rat. J. Pharmacol. Methods, 16: 251-259, 1986.[Medline]
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