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Research ArticleArticle
Deletion of the NADPH-Cytochrome P450 Reductase Gene in Cardiomyocytes Does Not Protect Mice against Doxorubicin-Mediated Acute Cardiac Toxicity
Cheng Fang, Jun Gu, Fang Xie, Melissa Behr, Weizhu Yang, E. Dale Abel and Xinxin Ding
Drug Metabolism and Disposition August 2008, 36 (8) 1722-1728; DOI: https://doi.org/10.1124/dmd.108.021881
Cheng Fang
Jun Gu
Fang Xie
Melissa Behr
Weizhu Yang
E. Dale Abel
Xinxin Ding
A genetic mouse model (designated cardiomyocyte-Cpr-null) with cardiomyocyte-specific deletion of the cytochrome P450 (P450) reductase (Cpr) gene was generated in this study. CPR protein levels, as well the enzyme activity of P450s, were greatly reduced in heart microsomes from the null mice compared with wild-type mice, whereas CPR expression in other organs remained unchanged. Nonetheless, homozygous null mice were normal in appearance, gross anatomy, tissue morphology, and general cardiac functional parameters, and there was no indication of embryonic lethality or premature mortality in contrast to the recognized role of CPR in embryonic development. Thus, this new mouse model should be useful for determination of the in vivo roles of cardiomyocyte CPR and CPR-dependent enzymes, including microsomal P450s, not only in the metabolism and toxicity of numerous xenobiotic compounds but also in cardiac pathophysiology. As a first application, we studied the role of cardiomyocyte CPR and CPR-dependent enzymes in doxorubicin (Dox)-mediated acute cardiotoxicity. Wild-type and null mice were treated with a single i.p. dose of Dox at 5, 10, or 20 mg/kg. The Dox treatment caused apoptosis and vacuolization in cardiomyocytes at the dose of 20 mg/kg and a significant increase in the levels of serum creatine kinase at 10 and 20 mg/kg in both wild-type and null mice. However, there was no significant difference in the extent of Dox-induced cardiac injury between the two strains; this lack of difference suggests that cardiomyocyte CPR and CPR-dependent enzymes do not play critical roles in the acute cardiotoxicity induced by Dox.
Cytochrome P450 (P450) enzymes metabolize numerous exogenous and endogenous compounds. Multiple P450 enzymes are expressed in the heart, where they may participate in the metabolism of therapeutic agents and environmental toxicants (Wang et al., 2002; Williams et al., 2003), as well as endogenous arachidonic acid (AA) (Wu et al., 1996). Cardiac P450 enzymes are believed to play critical roles in cardiac ischemia-reperfusion injury through either increases in the production of free radicals or else alterations in the metabolism of AA (Granville et al., 2004; Nithipatikom et al., 2004; Seubert et al., 2004). However, the proposed in vivo functions of endogenous cardiac P450s, either in xenobiotic metabolism and/or toxicity or else in cardiac pathophysiology, remain largely unconfirmed.
The activity of microsomal P450 enzymes is dependent on the participation of NADPH-P450 reductase (CPR), which provides electrons needed for P450-catalyzed monooxygenase reactions, as well as for the reactions catalyzed by several other enzymes, including heme oxygenases (HOs), cytochrome b5, squalene monooxygenase, and fatty acid elongases (cf. Gu et al., 2003). Mice with germline inactivation of the Cpr gene die at the embryonic stage as a result of abnormal development of the brain, eye, heart, and limbs (Shen et al., 2002). Recently, transgenic mouse strains in which the Cpr gene was flanked by two loxP sites (designated Cprloxmouse) were generated (Wu et al., 2003); this genetic engineering permits tissue-specific Cpr knockout in postnatal mice.
To facilitate efforts to determine the biological functions of cardiac P450 enzymes in adults, as well as the roles of cardiac CPR and CPR-dependent enzymes in drug-mediated cardiotoxicity, we have now generated a cardiomyocyte-specific Cpr-null mouse model, designated the cardiomyocyte-Cpr-null mouse. This new mouse model was generated by cross-breeding the Cprlox mouse with a transgenic mouse (named αMHC-Cre) that affords cardiomyocyte-specific expression of the Cre recombinase (Cre) (Abel et al., 1999). In the αMHC-Cre mouse, the expression of Cre was driven by the promoter of the α-myosin heavy chain gene. The new cardiomyocyte-Cpr-null mouse was characterized for potential abnormality in appearance, gross anatomy, heart morphology, and heart weight; potential occurrence of embryonic lethality or premature mortality; potential changes in general cardiac function; and potential compensatory expression of microsomal P450s and HOs. As the first application of this new mouse model, we further determined whether the null and wild-type mice differ in their sensitivities to toxic doses of doxorubicin (Dox), a widely used anticancer drug; our purpose was to assess whether target-tissue CPR/P450-mediated Dox metabolism plays an important role in Dox-induced acute cardiotoxicity.
The clinical use of Dox is often limited by the dose- and time-dependent Dox-mediated acute and chronic cardiotoxicity (Singal and Iliskovic, 1998; Menna et al., 2008). Although the mechanisms of Dox-mediated cardiotoxicity are not fully understood, oxidative stress seems to play an important role, as indicated by the occurrence of cardiac lipid peroxidation (Myers et al., 1977), glutathione depletion (Doroshow et al., 1981), and decreases in glutathione peroxidase expression and activity (Li and Singal, 2000) in Dox-treated animals. Dox-induced oxidative stress and cardiotoxicity can be alleviated by pretreatment of animals with antioxidants, such as N-acetylcysteine and probucol (Doroshow et al., 1981; Li and Singal, 2000). Dox can undergo one-electron reduction by one of several enzymes, including the CPR; the reaction produces a semiquinone free radical, which can either directly attack cellular macromolecules or else indirectly (through reactions with oxygen and redox cycling) yield superoxide anion, hydrogen peroxide, and hydroxyl radicals, with consequent cellular damage to the cardiac tissue (Xu et al., 2005). Biotransformation of Dox in humans involves primarily the formation of doxorubicinol in a reaction catalyzed by carbonyl reductase, although Dox can also be metabolized by CPR through reductive deglycosylation to a nontoxic metabolite (Niitsu et al., 2000; Riddick et al., 2005). The critical role of carbonyl reductase in Dox-induced cardiotoxicity has been shown with transgenic and knockout mouse models (Forrest et al., 2000; Olson et al., 2003); nevertheless, the in vivo function of CPR and P450 in Dox-mediated cardiotoxicity remained to be shown. Our findings establish that cardiomyocyte CPR and CPR-dependent enzymes do not play critical roles in the acute cardiotoxicity induced by Dox.
Generation of the Cardiomyocyte-Cpr-Null Mice. Heterozygous αMHC-Cre mice on the FVB genetic background (Abel et al., 1999) were first crossed with Cprlox/lox mice on a mixed C57BL/6 and 129/Sv background (Wu et al., 2003); the resultant αMHC-Cre+/-/Cprlox/+ progeny were crossed with Cprlox/lox mice again, yielding pups with one of four possible genotypes: αMHC-Cre+/-/Cprlox/lox (null) and αMHC-Cre-/-/Cprlox/lox (wild-type littermate), as well as αMHC-Cre+/-/Cprlox/+ and αMHC-Cre-/-/Cprlox/+ (neither of the latter two was used for this study). Cre and Cpr genotypes were determined by polymerase chain reaction analysis of tail DNA, as previously described (Wu et al., 2003). All the animal studies were approved by the Institutional Animal Care and Use Committee of the Wadsworth Center.
Immunoblot Assays. Protein samples were fractionated on 10% polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). For immunodetection, the following rabbit or goat antibodies were used with the indicated dilution: anti-CPR (1:1000; Stressgen, San Diego, CA), anti-HO-1 (1:500; Stressgen), anti-HO-2 (1:5000; Stressgen), anti-CYP1A1/2 (1:750; BD Gentest, Woborn, MA), anti-CYP2B1/2 (1:1000; BD Gentest), anti-human CYP2C9 (1:1000; BD Gentest), anti-human CYP2E1 (1:1000; Oxford Biomedical Research, Oxford, MI), anti-CYP3A2 (1:1000; BIOMOL International, Plymouth Meeting, PA), and anti-CYP4A (1:1000; BD Gentest). All the antigens were derived from the rat unless indicated otherwise. The blots were analyzed with use of an enhanced chemiluminescence kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK), and the signal intensity was quantified with a densitometer.
Tissue Preparation. Mice were sacrificed by CO2 asphyxiation. For histological analysis, hearts were dissected, fixed in 10% formalin for 24 h, embedded in paraffin, and cut into 5-μm sections at the coronal plane. Sections were stained with hematoxylin and eosin (H&E). For immunohistochemical analysis, mice were first perfused with 20 ml of phosphate-buffered saline (PBS), pH 7.4, followed by 50 ml of freshly prepared, ice-cold 4% paraformaldehyde in PBS. The hearts were dissected and postfixed in 4% paraformaldehyde for 2 h. Cryoprotection was conducted by placing fixed hearts in 30% sucrose in PBS for 24 h. The hearts were then flash-frozen in TissueTek OCT (Sakura Finetek, Torrance, CA) and sectioned at 10 μm with a cryostat (Sakura Finetek). Frozen sections were mounted on Superfrost Plus microscope slides (Fisher Scientific, Springfield, NJ) and stored at -80°C until use.
Immunohistochemical Analysis. Frozen sections were brought to room temperature for 30 min and rehydrated using PBS, followed by incubation with Triton X-100 (0.3% in PBS) for 30 min, to increase the permeability of the tissue sections. Nonspecific sites on the sections were blocked by two sequential incubation steps, first with Image-iT FX signal enhancer (Molecular Probes, Eugene, OR) for 30 min and then with a blocking solution (3% bovine serum albumin/0.1% Triton X-100 in PBS) for 1 h. Sections were incubated with rabbit anti-CPR antibody (Stressgen, diluted at 1:100 in the blocking solution) overnight at room temperature; then, after washing with PBS for 30 min, they were incubated with Alexa-594-conjugated chicken anti-rabbit antibody (1:200 dilution; Molecular Probes) for 1 h at room temperature. Sections were mounted with Prolong Gold antifade reagent with 4,6-diamidino-2-phenylindole (DAPI) (Molecular Probes). The primary antibody was omitted for negative control slides. Sections were observed using a Nikon Eclipse i50 fluorescence microscope (Nikon, Tokyo, Japan).
Isolation of Cardiomyocyte. Ventricular cardiomyocytes were isolated according to a previously reported method (Cheng et al., 2004). Briefly, adult mice were injected with heparin (100 U/mouse) 30 min before surgical operation. Mice were anesthetized with a mixture of ketamine (10 mg) and xylazine (0.7 mg). The heart was quickly removed, placed in ice-cold perfusion buffer (Cheng et al., 2004), and then cannulated to a 22-gauge needle with a blunt tip. Perfusion was started immediately at a constant flow rate (3 ml/min) with prewarmed perfusion buffer (37°C). After perfusion for 3 to 4 min, the perfusion buffer was replaced with prewarmed (37°C) digestion buffer, which consisted of the perfusion buffer containing 0.25 mg/ml Liberase Blendzyme I (Roche, Indianapolis, IN), 0.14 mg/ml trypsin (Invitrogen, Carlsbad, CA), and 12.5 μM CaCl2. After 10–12 min, the perfusion with digestion buffer was stopped. The heart was cut into pieces of ∼1 mm3 in size in a 60-mm cell culture dish. Tissue pieces were pipetted 5 to 10 times with a wide-opening plastic pipette, and they were then quickly transferred to a 15-ml tube containing 5 ml of a stopping buffer (perfusion buffer containing 10% calf serum and 12.5 μM CaCl2). Cells in the tissue pieces were dissociated by gentle pipetting; the resulting cell suspension was filtered through a metal sieve (300-μm mesh), and the cells were allowed to settle by gravity for 10 to 15 min. The supernatant was decanted, whereas the cell pellet was collected and stored at -80°C until use.
Measurement of Microsomal P450 Activity. P450-catalyzed dealkylase activity was measured for cardiac microsomes with a sensitive fluorescence-based method (Ekins et al., 1998). The substrate, 3-cyano-7-ethoxycoumarin (nonfluorescent), can be dealkylated by several P450 enzymes, including CYP1A1, CYP1A2, and CYP2B1, to form 3-cyano-7-hydroxycoumarin, a fluorescent metabolite (White, 1988). Cardiac microsomes were prepared from pooled ventricles (from five to seven adult mice for each group). The reaction was initiated by the addition of 2.0 mg of freshly prepared microsomal protein to a mixture containing 400 μl of an NADPH-regeneration system (Zhuo et al., 2004), 2 μl of 3-cyano-7-ethoxycoumarin (Molecular Probes; dissolved in dimethyl sulfoxide) for a final concentration of 9.3 μM, and water to give a final reaction volume of 1.0 ml. After 45 min at 37°C, with constant shaking, the reaction was stopped by the addition of 2.0 ml of acetonitrile. The mixture was centrifuged at 12,000g for 5 min. The supernatant was filtered through a 13-mm syringe filter (0.45 μm in diameter). Product fluorescence was measured using a PerkinElmer Life and Analytical Sciences (Boston, MA) LS50B Luminescence Spectrometer, with excitation at 408 nm (slit width 5 nm) and emission at 450 nm (slit width 10 nm). Standard curves were prepared with authentic 3-cyano-7-hydroxycoumarin (Molecular Probes). The rates of product formation were linear with incubation time, up to 90 min under the reaction conditions described above.
Doxorubicin-Induced Acute Cardiac Toxicity. Two-month-old male null and wild-type mice were injected i.p. with doxorubicin hydrochloride (Sigma, St. Louis, MO) at 5, 10, or 20 mg/kg (n = 5 in each group), whereas the control null and wild-type groups were injected with saline (n = 4). Four days after injection, mice were sacrificed with CO2, and blood samples were collected from the right ventricle. Paraffin sections of the hearts were stained with H&E for histological examination.
Analysis of Apoptosis in Mouse Heart. Cardiomyocyte apoptosis was analyzed in situ by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, with the ApopTag Fluorescein in Situ Apoptosis Detection Kit (Chemicon, Temecula, CA). The assay was conducted with 4-μm paraffin sections according to the manufacturer's instructions, with DAPI as a counterstain. Sections of rat mammary gland (at 4 days after weaning of her pups) were used as positive controls for apoptosis. Three-month-old male null and wild-type mice were injected i.p. with doxorubicin at 20 mg/kg (n = 4 in each group), whereas the control null and wild-type groups were injected with saline (n = 4). Two days after the treatment (Liu et al., 2005), mice were sacrificed by CO2 asphyxiation and transcardially perfused, sequentially, with PBS and 10% formalin solution. The heart was removed and immersed in 10% formalin for 24 h; it was then halved, processed, and embedded in paraffin block for tissue sectioning. The total number of apoptotic cells per section was counted under a fluorescence microscope with a fluorescein isothiocyanate filter (Nikon Eclipse i50). Concurrently, the digital image of each section was taken by a fluorescence stereomicroscope (Olympus SZX12, Olympus, Tokyo, Japan) with DAPI/UV filter, with the area of section measured by SPOT software version 4.6 (Diagnostic Instruments, Sterling Heights, MI). The index of apoptosis was expressed as the number of apoptotic cells per millimeter squared. For each heart, two sections were quantified, and the results were averaged.
Other Methods. Serum creatine kinase (CK) activity was measured with a commercial kit (Diagnostic Chemicals, Oxford, CT) using 40 μl of serum for each measurement. A noninvasive blood pressure acquisition system (CODA 6; Kent Scientific, Torrington, CT) was used for measuring parameters of cardiac function. Systolic blood pressure, diastolic blood pressure, mean blood pressure, and heart rate were measured for conscious, 2-month-old, male null and wild-type mice (n = 5 in each group). Microsomes from the heart and other tissues were prepared according to previously described methods (Gu et al., 2003). Protein concentration was determined by the bicinchoninic acid assay (Pierce, Rockford, IL).
Cardiomyocyte-Specific Cpr Deletion. Tissue-specific deletion of the Cpr gene in the cardiomyocyte-Cpr-null mouse was shown by immunoblot analysis of microsomal preparations from various tissues of adult null mice and their wild-type littermates. As shown in Fig. 1A, for 2- to 4-month-old male mice, microsomal CPR protein levels were decreased in both the ventricular and atrial samples from the null mice compared with age-matched wild-type controls. Densitometric analysis indicated that the extents of decrease in CPR protein expression in the null mice were 70 and 50% in ventricular and atrial samples, respectively (Fig. 1B). The occurrence of significant levels of residual CPR protein in the null mice was not unexpected, given the fact that CPR and/or P450 are known to be expressed not only in cardiomyocytes but also in cardiac fibroblast and endothelial cells (Brittebo, 1994; Borlak et al., 2003). In experiments not shown, CPR activity toward cytochrome c was detected in ventricular microsomes from wild-type, but not in those from the null, mice; however, the low overall rates detected did not permit an accurate determination of the extent of decrease in the null mice. In contrast, the levels of CPR protein were not significantly changed in other organs examined, including liver, lung, kidney, and brain (Fig. 1C).
As expected, deletion of the Cpr gene in the cardiomyocyte was permanent, as indicated by the fact that a decreased CPR expression was found for cardiac microsomal samples from 13-month-old null mice compared with age-matched wild-type littermates (data not shown). To confirm the occurrence of Cpr deletion in cardiomyocytes, we compared CPR protein levels in isolated cardiomyocytes from adult null and wild-type mice. Microscopic inspection of the isolated cell preparations indicated minimal contamination by cardiac fibroblasts (data not shown). As shown in Fig. 1, A and B, the level of microsomal CPR protein in the isolated cardiomyocytes was ∼85% lower in the null mice than that in wild-type littermates; this extent of decrease in CPR expression, which may partly reflect the presence of contaminating CPR-expressing cardiac fibroblasts, is similar to the known extents (80–90%) of Cre-mediated gene recombination in cardiomyocytes of other αMHC-Cre+ mice (Duan et al., 2005).
The cellular distribution of CPR expression in the hearts of the null and wild-type mice was analyzed by immunohistochemistry (Fig. 2). In the wild-type mice, CPR protein was detected in the cardiomyocytes, the cell type that makes up cardiac muscle fibers, and also in other cell types, including cardiac fibroblasts and endothelial cells. In the null mice, the intensity of immunoreactive signals in the cardiomyocytes was reduced to the level seen in the negative control section, whereas CPR staining in other cell types remained unchanged compared with the staining intensity in the wild-type mice (Fig. 2). These results further showed that cardiomyocyte-specific Cpr deletion was achieved in the cardiomyocyte-Cpr-null mice.
Immunoblot analysis of CPR expression in wild-type and cardiomyocyte-Cpr-null mice. A, immunoblot analysis of CPR expression in cardiac microsomes. CPR protein was detected with a polyclonal anti-rat CPR antibody. Microsomes were prepared from tissues pooled from 5 (for ventricle) or 10 (for atrium) 2- to 4-month-old male mice. For cardiomyocytes, the microsomes were prepared from cells isolated from six adult male mice. Each lane was loaded with 10 μg (for ventricle and cardiomyocytes) or 6 μg (for atrium) of microsomal protein. Samples were analyzed in duplicate. B, densitometric analysis of the CPR band detected in A. The data shown represent the ratios of the averaged band intensities for the null mice versus the intensity for the wild-type littermates. C, immunoblot analysis of CPR levels in microsomal preparations from liver, kidney, brain, and lung. Three microsomal preparations, each from one adult male mouse, were analyzed for each mouse strain. Each lane was loaded with 5 μg (for liver) or 10 μg (for the other tissues) of protein.
Immunofluorescent detection of CPR in the wild-type and cardiomyocyte-Cpr-null hearts. Frozen sections (10 μm thick) from 2-month-old male mice were incubated with a rabbit anti-CPR antibody. CPR expression was visualized by a chicken anti-rabbit secondary antibody conjugated with Alexa 594 (red fluorescence). The sections were counterstained with DAPI (blue fluorescence). The anti-CPR antibody was omitted in the negative control section. CPR immunostaining was found in cardiomyocytes (small arrowheads) and other cell types, including cells that appear to be cardiac fibroblasts (large arrowheads); bar = 50 μm. The results shown are typical of sections from seven mice for each strain.
General Characterization of the Cardiomyocyte-Cpr-Null Mice. The null mice were indistinguishable from the wild-type mice in gross appearance, daily behavior, body weight, and mortality rates at various ages, ranging from 2 to 18 months. The ratios of heart weight to body weight in the male (Table 1) and female (data not shown) null mice were not different from the ratios in wild-type littermates, indicating the absence of cardiac hypertrophy. Similarly, no difference was found in the weights of other major organs, relative to body weight, between the null and the wild-type mice (data not shown). Hemodynamic parameters of the adult mice, including systolic blood pressure, diastolic blood pressure, and heart rate, did not show significant difference between the wild-type and the null mice (Table 1). These results indicated that the hearts of the null mice are functionally normal. In addition, both male and female null mice were fertile, and an analysis of genotype distribution among pups resulting from intercrossed heterozygotes indicated no sign of embryonic lethality (data not shown).
Parameters of cardiac function in cardiomyocyte-Cpr-null mice Body and heart weights were measured in 2-month-old male mice (n = 10–11 for each strain). Systolic and diastolic blood pressure and heart rate were measured by a noninvasive blood pressure acquisition system (n = 4–5 for each strain). The values presented are mean ± S.D. There was no significant difference between the wild-type and null groups for any of the parameters measured (P > 0.05, Student's t test).
Impact of Cardiomyocyte Cpr Deletion on Cardiac Microsomal P450 Activity. Cardiac microsomes prepared from pooled ventricles from five to seven adult wild-type or null mice were assayed for activity in the O-deethylation of 3-cyano-7-ethoxycoumarin. With the substrate at 9.3 μM, the initial rates of 3-cyano-7-hydroxycoumarin formation for the null mice (0.09 ± 0.06 pmol/min/mg protein; mean ± S.D., n = 4) were significantly lower (P < 0.05) than the rates found for the wild-type mice (0.16 ± 0.07 pmol/min/mg protein; n = 4), a result indicating decreases in cardiac microsomal P450 activity concomitant with decreases in the level of CPR protein.
Impact of Cpr Deletion on Cardiac P450 and HO Expression in the Null Mice. Given the previously reported compensatory gene expression changes in the liver of the liver-Cpr-null mice (Gu et al., 2003; Henderson et al., 2003), we performed immunoblot analysis to detect potential compensatory increases in the levels of CPR-dependent enzymes, including P450 and HO, in the hearts of the cardiomyocyte-Cpr-null mice. As shown in Fig. 3, small increases in the levels of CYP1A1/2 and HO-2 proteins, but no increases in the levels of CYP2C, CYP2J, and CYP3A proteins, were observed in ventricular microsomes from the null mice compared with the levels in the wild-type mice. Densitometric analysis (not shown) of the immunoblots revealed that the extent of increase was ∼40% for both CYP1A1/2 and HO-2. The cardiac microsomal levels of CYP2B and HO-1 proteins were near the detection limit for both mouse strains, thus making it difficult to compare the relative expression levels between the two groups. The expression of CYP2E1 and CYP4A was not detected in either type of microsomal sample (data not shown).
Impact of Cardiomyocyte Cpr Deletion on Dox-Induced Acute Cardiotoxicity. The extent of Dox-induced acute cardiotoxicity was determined by three different indices: serum levels of CK, numbers of apoptotic cells, and degrees of histological abnormality. The serum levels of CK activity were determined for adult, male wild-type and null mice at 4 days after treatment of the animals with a single i.p. injection of Dox (at 5, 10, or 20 mg/kg, respectively) or saline. Dox treatment caused a dose-dependent increase of the CK levels in both wild-type and null mice compared with saline-treated groups; however, there was no significant difference in the CK levels either between saline-treated wild-type and null mice or between Dox-treated wild-type and null mice at any of the tested dosages (Fig. 4); the latter result is indicative of similar extents of Dox-induced acute cardiotoxicity in the two mouse strains.
Apoptosis in the heart was analyzed by TUNEL assay in adult, male wild-type and null mice at 2 days after treatment with doxorubicin (at 20 mg/kg) or saline. An earlier time point (2-day), compared with the time point chosen for CK determination and histological analysis (4-day), was selected based on previous reports indicating that cardiomyocyte apoptosis is an early event after Dox treatment (Arola et al., 2000). The numbers of apoptotic cells per millimeter squared of tissue section in saline-treated wild-type and null groups were similar, at ∼1/mm2 (Fig. 5). Dox treatment caused a 3-fold increase in apoptotic cells in both wild-type and null groups (P < 0.01, versus saline-treated wild-type and null groups, respectively). However, there was no significant difference between Dox-treated wild-type and null mice.
Histological analysis of H&E-stained heart sections from saline-treated mice indicated that the hearts of wild-type and null mice are indistinguishable (Fig. 6, A and B). At 4 days after Dox treatment, hearts from both wild-type and null mice showed widely scattered foci of one to several cardiomyocytes, with loss of striations, and granular, fragmented cytoplasm or cytoplasmic vacuolization. These foci of myocardial degeneration, which were absent in saline-treated mice, were found in ventricular and atrial myocardium in the Dox-treated mice from both strains (Fig. 6). These results further indicate that the deletion of Cpr in the cardiomyocytes does not protect the hearts from Dox-induced acute toxicity.
The impact of cardiomyocyte-specific Cpr deletion on cardiac P450 and HO expression. Microsomes prepared from pooled ventricles from five adult male mice were analyzed for each strain. Samples were loaded in duplicate. Each lane contained 10 μg of protein. The antibodies used are described under Materials and Methods.
Dox-induced increase in serum CK activity. Three-month-old male mice were given a single i.p. injection of Dox (at 5, 10, or 20 mg/kg; n = 5) or saline (n = 4). Four days after Dox treatment, serum samples were obtained, and CK activity was determined as described under Materials and Methods. The enzyme activity (rate of formation of NADPH) was expressed as units per liter, which was calculated as [changes in absorbance per minute] × 4180. *, P < 0.05; **, P < 0.01, compared with saline-treated mice of the same strain (Student's t test).
Dox-induced cardiac apoptosis. Three-month-old male mice were given a single i.p. injection of Dox (at 20 mg/kg; n = 5) or saline (n = 4). Two days after treatment, apoptotic cells were detected by TUNEL assay in paraffin-embedded heart sections as described under Materials and Methods. **, P < 0.01, compared with saline-treated mice of the same strain (Student's t test).
Dox-induced cardiac pathologic changes. Hearts were obtained 4 days after Dox (at 20 mg/kg) or saline treatment. Coronal sections of the heart were stained with H&E. A, saline-treated wild-type mice; B, saline-treated null mice; C, Dox-treated wild-type mice; D, Dox-treated null mice. The results shown are typical of four mice examined in each group. Arrows indicate sites of myocardial degeneration; bar = 50 μm.
In this study, we have generated and characterized a mouse model with cardiomyocyte-specific deletion of the Cpr gene. The cardiomyocyte-Cpr-null mouse joins a growing list of conditional knockout mouse models that target the Cpr gene; these models can have valuable research applications in drug metabolism and toxicology. For examples, Cpr was deleted specifically in hepatocytes in liver-specific Cpr-null mouse models through the use of an albumin-Cre transgenic mouse. Studies using the liver-Cpr-null mice, in which the liver microsomal CPR levels were reduced by ∼95% from wild-type levels, revealed significant roles of hepatic CPR-dependent enzymes not only in cholesterol biosynthesis but also in systemic xenobiotic disposition and hepatic chemical toxicity (Gu et al., 2003). In a more recent study, a lung-Cpr-null mouse model was generated, in which Cpr was specifically deleted in a large fraction of type II alveolar epithelial cells and Clara cells (Weng et al., 2007). Studies using the lung-Cpr-null mouse indicated that pulmonary P450-mediated metabolic activation plays an essential role in the lung tumorigenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco-specific carcinogen.
In the present study, we find that cardiomyocyte CPR is dispensable for cardiac function in adult mice, given that the cardiac histology and hemodynamics seen in the cardiomyocyte-Cpr-null mice were normal. This result was in contrast to the documented critical function of CPR during embryonic development. Germline deletion of Cpr caused embryonic death in midgestation, accompanied by severe structural defects in multiple fetal organs, including the heart (Shen et al., 2002). In our cardiomyocyte-Cpr-null model, the expression of Cre, controlled by the promoter of αMHC, is expected to occur as early as E8.5, and Cre-mediated homologous recombination in the cardiomyocytes can take place at E11.5 (Gaussin et al., 2002). The functional impact of the Cpr deletion on embryonic metabolism of endogenous P450 substrates, such as retinoic acids, may not be significant until E13 or later, when sufficient amounts of the pre-existing CPR protein in targeted cardiac cells have been degraded. By then, the role of CPR and CPR-dependent enzymes in cells targeted for Cpr deletion may be no longer critical for development. Alternatively, the metabolic deficiency in the Cpr-null cells could be overcome by surrounding cells with normal CPR expression; for example, retinoic acids accumulated in the Cpr-null cells could be transported to neighboring Cpr+ cells for P450-mediated degradation. Notably, no developmental or structural defect was found in the lungs of our recently reported lung-Cpr-null mouse model, in which Cre-mediated Cpr deletion was also expected to occur in mid to late gestation (Weng et al., 2007).
The cardiomyocyte-Cpr-null model can be used to determine the biological functions of cardiomyocyte microsomal P450 enzymes in the heart. Although the overall level of P450 expression in the heart is quite low compared with the level in the liver and other portal-ofentry organs, specific P450 enzymes, such as CYP2J and CYP2C, are relatively abundant in the cardiovascular system, where they are thought to regulate various biological and/or pathological processes through the metabolism of pertinent endogenous substrates (Wu et al., 1996; Enayetallah et al., 2004). For example, a number of P450 enzymes, including CYP2Js and CYP2Cs, can metabolize AA to epoxyeicosatrienoic acids and hydroxyeicosatetraenoic acids (Fleming, 2001). The epoxyeicosatrienoic acids could play a protective role by dilating coronary arteries during ischemia and reperfusion (Campbell and Harder, 1999) as shown by findings from a cardiomyocyte-specific CYP2J2-transgenic mouse, in which microsomal AA epoxygenase activity was 3-fold higher than in the wild-type mouse and in which postischemic recovery of the left ventricular function was significantly improved (Seubert et al., 2004). On the other hand, 20-hydroxyeicosatetraenoic acid seems to play a major role in exacerbating cardiac postischemic damage (Nithipatikom et al., 2004). Nonetheless, definitive evidence has yet to be obtained for a role of endogenous P450 enzymes of the cardiomyocyte, as opposed to enzymes of cardiac vascular tissues (Enayetallah et al., 2004; Delozier et al., 2007) or elsewhere, in the postischemic cardiac injury. This knowledge gap can now be filled at least partly through studies using the cardiomyocyte-Cpr-null mice. In this context, we recently described another transgenic mouse model, termed the Cpr-low (or Cprlow) mouse, in which the expression of CPR was greatly decreased (although not abolished) in all the organs and cell types examined (Wu et al., 2005). In the hearts of the Cpr-low mice, CPR expression at 2 months of age was decreased by ∼90% compared with wild-type C57BL/6 and 129/Sv mice. The combined use of the Cpr-low and the cardiomyocyte-Cpr-null mouse models will likely be useful for distinguishing the relative roles of cardiomyocytes and the cardiac vascular system in postischemic reperfusion injury.
Findings of previous in vitro studies had suggested that CPR is involved in Dox-mediated cardiotoxicity through its catalysis of a one-electron reduction of Dox; the semiquinone radical formed in this process can induce increased oxidative stress through the generation of superoxide anion and then hydrogen peroxide and reactive hydroxyl radicals (Riddick et al., 2005; Xu et al., 2005). However, the results from the current in vivo study do not support the notion that CPR plays a major role in Dox-mediated acute cardiotoxicity. Besides CPR, there are several other enzymes, including flavin reductase, NADH dehydrogenase, and xanthine oxidase, that could catalyze the one-electron reduction of Dox (Doroshow, 1983). The activities of these other redox enzymes in the cardiac tissue appear to be sufficiently high for mediation of Dox-induced toxicity even in the absence of contributions from CPR. Notably, our finding is consistent with that of Ramji et al. (2003), who studied the role of CPR in Dox-induced toxicity in cultured cells. They showed that, although microsomal CPR activities in CPR-overexpressing human lymphoblastoid cells and human liver microsomes correlated well with the rates of Dox reduction, the susceptibility of human breast cancer cells to Dox-mediated cytotoxicity could not be enhanced through overexpression of CPR (Ramji et al., 2003). However, it remains to be determined whether CPR plays a major role in Dox-induced chronic cardiotoxicity when animals are treated continually with the drug at doses lower than those used in the present study; the various enzymes capable of forming the Dox semiquinone radical may differ in their enzyme kinetic parameters with respect to Dox or in their propensity to be inactivated by the oxidants produced in Dox-induced redox cycling.
It will also be interesting to determine whether the Cpr-low mouse model is protected against Dox-induced acute and/or chronic cardiotoxicity, given the potentially important role of CPR in the cardiac vascular system. In the Cpr-low mouse, the contribution of CPR to Dox-mediated toxicity would be substantially decreased in both cardiac muscles and cardiac vasculature. In that regard, although CPR expression is also decreased in the liver and elsewhere in the Cpr-low mice, systemic Dox clearance is unlikely to differ between the Cpr-low mice and wild-type mice. This assumption is supported by findings of a recent study using a liver-specific Cpr-null mouse model; there, hepatic CPR/P450 did not play a critical role in Dox clearance (Henderson et al., 2006).
In summary, we have generated and characterized a transgenic mouse model with cardiomyocyte-specific Cpr deletion. The null mouse exhibits normal tissue morphology and cardiac function. An initial application of this mouse model led to the finding that cardiomyocyte CPR and CPR-dependent enzymes do not play a critical role in the acute cardiotoxicity induced by Dox in vivo. This new knockout mouse model will be valuable for exploring potential functions of cardiomyocyte P450 enzymes in various physiological and pathological processes, such as postischemic reperfusion injury and xenobiotic-induced cardiotoxicity.
We thank the Pathology Laboratory, as well as the Biochemistry and Molecular Genetics Core Facilities, of the Wadsworth Center for the use of their services. We also thank Dr. Adriana Verschoor for reading the manuscript.
This study was supported in part by National Institutes of Health Grants ES07462 (to X.D.) and HL70070 (to E.D.A., who is an established investigator of the American Heart Association).
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.108.021881.
ABBREVIATIONS: P450, cytochrome P450; AA, arachidonic acid; CPR, cytochrome P450 reductase; HO, heme oxygenase; Cre, Cre recombinase; Dox, doxorubicin; H&E, hematoxylin and eosin; PBS, phosphate-buffered saline; DAPI, 4,6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; CK, creatine kinase.
Received April 15, 2008.
Accepted May 5, 2008.
The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition August 1, 2008, 36 (8) 1722-1728; DOI: https://doi.org/10.1124/dmd.108.021881
Identification of payload-containing catabolites of ADCs
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The Atkins Food plan is the best made her fritters growing up and its how my family.
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Your treatment journey- what comes next?
Your colorectal cancer survivorship journey doesn't necessarily end once you have received a clean bill of health. Dr. Lidia Schapira (of the American Society of Clinical Oncology) explains some of the things you may feel or experience once you've beat the disease. Know what to expect post treatment, and don't be afraid to ask for help if you need it.
Remember that the CCAC will continue to be here for you, whether you have beaten cancer yourself or helped a loved one conquer it. Many survivors find their journey enriched by helping others. Becoming a CCAC Cancer Coach is a way you can bring support into your own community; many of our dedicated program volunteers have beaten the disease themselves and have gone on to inspire hope in others.
Thank you to @LibbyShope on Twitter, for making us aware of this video.


text:
This October 15, 2018, GOLD image of daytime airglow clearly shows the equatorial ionization anomaly (EIA) as two arcs on either side of the magnetic equator, which extend across the nightside of the disk. The colors represent an increase in oxygen emissions from blue to red, with the left side of the Earth lit by the Sun, which has set over most of South America. (Courtesy GOLD)
NASA's Global-scale Observations of the Limb and Disk (GOLD) mission has observed dramatic and unexplained shifts in the location of features in the Earth's ionosphere surrounding the equator. Unanticipated changes in the nighttime ionosphere can lead to disruptions in communication and navigation that depend on satellites, such as GPS.
GOLD is an ultraviolet imaging spectrograph that was designed and built at the University of Colorado Boulder's Laboratory for Atmospheric and Space Physics (LASP) and is hosted on the SES-14 communications satellite. The latest discoveries from the mission are challenging mission scientists and were published last week in Geophysical Research Letters.
Since reaching orbit in October 2018, GOLD has been making observations of the Equatorial Ionization Anomaly (EIA), regions of the ionosphere with enhanced electron density north and south of the magnetic equator. One of the primary goals of the mission is to better understand the behavior of the EIA and the instabilities within it. GOLD presents a new ability to image the variability of ionospheric plasma and, ultimately, to understand its causes.
"The variability of the nighttime ionosphere has been a puzzle that scientists have studied for more decades than most of us have lived," said Richard Eastes, GOLD principal investigator and LASP research scientist. "Today it's an important problem to solve because better forecasts are needed for reliable navigation and communication by airplanes and others."
Density variations in the ionosphere can cause localized plasma depletions called "bubbles." Radio waves, including those used for communications and navigation, can be disrupted when they encounter these bubbles. The occurrence of bubbles and other ionospheric irregularities presents a critical challenge for efforts to predict how changes in solar and geomagnetic activity affect the nighttime ionosphere.
Because of GOLD's unique perspective from geostationary orbit and its rapid imaging cadence (a complete scan of each hemisphere every 15 minutes), GOLD is providing new information that has not been available in previous EIA observations. From its geostationary vantage some 22,000 miles above the Earth, GOLD observes the same longitudes repeatedly every night and provides large scale images of the EIA.
GOLD has observed gaps occurring on a majority of the nights and has more irregularly detected abrupt changes in the location of the EIA. The team will rely on additional measurements before attempting to determine the cause of variability in the EIA and its changing location.
GOLD is a NASA mission of opportunity managed by the Goddard Space Flight Center in Greenbelt, MD. The GOLD principal investigator is based at LASP, which built the instrument, provides project management and systems engineering, as well as instrument operations for the GOLD mission. GOLD is a hosted payload on a commercial communications satellite, SES-14, built by Airbus for Luxembourg-based satellite operator, SES.
AGU, EIA, Equatorial Ionization Anomaly, Geophysical Research Letters, GOLD, GPS, GRL, NASA, NASA Goddard, SES-14


The BEAN: What does a logo say about BEA?
What does a logo say about BEA?
The BEA site is freshly dressed with BookExpo America's new branding and logo. While the new BEA logo will look nice on a t-shirt, it won't do a thing for any exhibitor or attendee. What is tangible is that the new look of BEA is indicative of the transformation that is being applied to BEA from top to bottom. Some changes are old news with the fact that BEA is moved to mid-week (Tues. - Thurs) and the ABA will be doing their day of education at Javits and not offsite (plus ABA hotel will be in Manhattan, not Brooklyn, which may be a hipper, but not as easy for events during BEA).
The new BEA logo is fresh, simple and defined in the tag-line: The Content and The Buzz, which is what BEA needs to be for all of our participants. For our prized attendees - BEA will be easier to navigate both from the website and at BEA with more platforms to see, hear and interact with authors (a new author search tool will go live in March). For exhibitors, BEA will deliver more quality attendees, create more platforms for authors, there will be more relevant bloggers engaged and the inclusion of the IDPF Digital Book 2010 will bring digital solutions, reading devices and technology to BEA.
The new look and logo articulates that BEA is looking forward proactively to meet the needs of the entire publishing industry. Sharing a recent converstation evaluating BEA's prospects for 2010 - I am optimistic opposed to be hopeful. I am optimistic knowing that the changes BEA has invested in will deliver what the industry needs from BEA and I am not having to hope for any thing that is out of our control.
I look forward to sharing a number of smaller wins that I will be able to confirm in the next few weeks that will welcomed news to the activity happening at BEA 2010.


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Gateway Classic Cars of Scottsdale is honored to be the consignment showroom within the GCC network to sell this absolutely astounding 1968 Dodge Charger, built in the pro street style by Muscle Car Restorations in Wisconsin. Theyre a well-known shop, particularly for Mopars. Starting with a rust-free California car purchased in 2001, the car was sent to the Midwest for its transformation, which MCR completed after six years and 2800 hours! All of the latest hardware was used to make this Charger one of the nicest (and nastiest) modified Mopars ever built. This 68 has been featured in several magazines and has even made an appearance at the Playboy Mansion.Under the hood lurks some serious power with a Mopar 528 crate Hemi engine that has been modified with Keith Black components. Transmitting all 640 ponies (650 ft lbs torque) to the narrowed, Detroit Locker-equipped 9 rear axle is a Tremec 5-speed transmission with gears selected by a modified original Hurst Pistol Grip shifter. Putting the power to the ground is a one-off set of Boyds custom wheels.One of the trick features on this muscular Dodge, among countless mods done on the car, is the toggle switch hidden under the LH part of the steering column. Flipped one direction, the horn is activated, flip it the other way, youve got Line-locks! This car also has 4 wheel Wilwood brakes, 5 point seat belts, trunk mounted fuel cell, custom dash with built in shift light and much more. To duplicate this car today would cost in excess of $250,000-plus. This babys ready to rock and has had all of the bugs worked out. It runs, shifts, handles, brakes, and performs like a new car. Youll love this car! Rememer, yesterdays thinkers are todays buyers. To see 100-plus pictures and a video of this 1968 Dodge Charger pro touring, please visit www.gatewayclassiccars.com. For more information or to schedule an appointment to view this vehicle in person, please call the Scottsdale showroom at 623-900-4884 or email us directly at scottsdale@gatewayclassiccars.com See more at: http://www.gatewayclassiccars.com/SCT/52/1968-Dodge-Charger
1951 Mercury Coupe
If you are looking for a completely custom lead sled, then this 1951 Mercury 2-Door is for you! These cars were originally available with a base purchase price of $1,945. This car has under gone a complete restoration that took 8 years to complete and the whole build was centered around the Art Morrison chassis that lays hidden underneath. This custom-built chassis allows this car to lay down on the ground the way it should while also giving it a smooth ride. One of the most obvious custom touches on this Mercury is the slammed stance, thanks to the RideTech air ride suspension. Complete with onboard air compressor you can raise or lower the stance of this car with a touch of a button. The second most obvious touch on this Mercury is the custom front grill. It helps give this 2-door a signature look that is unmistakable to any other cars on the road today. This Mercury is painted in a beautiful deep blue that complements every sweeping bodyline that has been professionally touched and crafted to seperate it from its stock counterparts. Adding to the sleek appearance of this 2-door is the chopped roof. All trim pieces have been shaved from this beauty as well the door handles, trunk and hood have been shaved to complete the look and open via electric motors. The two-tone brown interior includes custom sewn front and rear seats, a custom center console with Dakota digital gauges. Willwood disc brakes on all four corners help bring this sled to a stop with ease. The slammed body sits on four deep dish Schott custom wheels. The engine bay contains a power house called an LSX 454 that is fuel injected and looks just as great as it runs. Complete with air conditioning, power steering and power brakes this car drives like a dream and will turn heads no matter where you take it. You can view this 1951 Mercury Eight 2-Door Coupe in greater detail including HD pictures and an HD video of it running and driving at Gatewayclassiccars.com. If you are interested in purchasing this vehicle or have more questions regarding it please call us at (303) 872-4722 or email us at Denver@gatewayclassiccars.com.
1995 Mercedes E-Class
Farmingdale, NY
Ask for: Reid Branston
email: reid@motorcarclassics.com
Engine: I6 3.2L
1928 Ford Model A PHAETON TOURING CAR
Malone, NY
Ask for: Joe
email: cars@abclassics.com
Engine: 4 cyl. gas engine
Transmission: Manual 4 speed trans
REDUCED 1928 Ford Model A Phaeton
Car was restored years ago and now needs a cosmetic restoration, mainly paint needed. Auto Trunk on the rear.
No Need to do a full restoration on another one when there is a value like this one available. Great for events, parades, weddings or summer cruising!
Sold at AnB Classic Cars, �Where Classic Cars Find New Homes�.
http://www.anbclassics.net/inventory.aspx
Ad ID: 309
Ask for: Mario Visone
email: mariocaddy@yahoo.com
Engine: 383 ci
1964 Chrysler 300 Coupe. NO RUST! NO ROT! This was a California desert car and is loaded with options.
WORKING factory Air Conditioning, Power Windows, Power Steering, Power Brakes, Power Locks, Power Antenna, Power Seats!
This is a very RARE MATCHING NUMBERS BIG BLOCK CAR! This Chrysler has had one repaint in it's original white color. The interior, which includes factory bucket seats and console shift, is in great condition. Just a real nice driver that runs and drives GREAT.
Original build sheet, Original rear seat covers and original air cleaner included as well. Call for more details or for financing information.... 631-620-3330
Visit Hollywood Motors online at www.hollywoodmotorsusa.com to see more pictures of this vehicle or call us at 516-CAR-GUYS today to schedule your test drive.
1947 Cadillac Series 62
Ask for: Nashville, TN
email: nashville@gatewayclassiccars.com
Gateway Classic Cars of Nashville is proud to offer for sale this 1947 Cadillac Series 62 luxury coach. This elegant 1947 Cadillac is perhaps the finest example on the road today. In the restoration and modification of this fine automobile, no expense was spared. This 1947 Cadillac Series 62 exudes class and comfort. Slide behind the wheel and the ultra luxurious leather and well detailed interior make this a motoring experience like no other. However, dont let the plush accommodations inside and glass like paint fool you, this car rides like a new vehicle thanks to its one off custom chassis built by Team Whitt Customs. Sporting tubular control arms, coil over suspension, disc brakes, and rack and pinion power steering this car maintains is timeless looks, while gaining the ease of driving and safety of these modern components. This 1947 Cadillac Series 62 is powered by a modern fuel injected LS 5.7L V8 backed by a 700R4 automatic transmission, providing excellent power and reliability for this superbly built motor vehicle. The car, features a vintage air unit, which provides ample heating and cooling, perfect for touring or for passengers as a coach for hire. This vehicle would be the perfect statement piece for a collector, or as an event vehicle for any upscale venue looking to add class and style to its fleet. Options include: Ac/Heat, USB input, Bluetooth, AM radio, wireless disabling device, power brakes, power steering, leather interior, disc brakes. This 1947 Cadillac Series 62 our Nashville showroom can be seen in greater detail including 100+ HD pictures and an HD video at www.gatewayclassiccars.com for more information please call the showroom at 615-213-1800 or email us at nashville@gatewayclassiccars.com if you would like to see this car in person we welcome you to stop by our Nashville showroom at 320 Tech park Dr. Suite 600 La Vergne Tn 37086.
1972 Pantera
Gateway Classic Cars of Philadelphia is offering this 1987 Pontiac Trans Am for sale. This Trans Am is finished in Dark Red with a Gray cloth interior. It is equipped with a 5.0 fuel injected V8 with an automatic transmission. This car has 18,180 actual miles and is in excellent condition. Equipment includes air conditioning, power steering and brakes, and power windows and locks. Cruise and tilt and an AM FM with cassette as well. This Trans Am also has the most popular option of all T TOPS. You just can't buy a Trans Am that doesn't have T TOPS. The 80's Trans Ams were one of the most popular cars of their day. The contemporary styling was ahead of its time and they held their own performance wise. It was one of the most aggressive designs to come out of Detroit at that time. To view this 1987 Pontiac Trans Am in greater detail, including HD pictures and an HD video, visit www.GatewayClassicCars.com or our Philadelphia showroom located in West Deptford, NJ. For more information, please call 856-599-4100 or email Philadelphia@GatewayClassicCars.com
Gateway Classic Cars of Chicago is proud to offer a super clean 2007 Ford Mustang GT. Finished in a lovely performance white, this GT coupe has a great look. It sits on premium 18 alloy wheel with BFGoodrich performance tires. To help accent the beautiful bright white exterior is the sporty red leather upholstery. The car has all the equipment youd expect in a modern-day muscle car including premium sound system, power windows, keyless entry, A/C and cruise control. Of course, performance enthusiasts are more concerned with whats under the hood: a 4.6 Liter 3 valve V8. In stock form the 4.6 is good for 300 horsepower and 320 lb ft of torque. To let the V8 breathe a bit, this one has a K&N cold air intake as well as a Pypes exhaust system which free up a few horses as well as making the car sound mean. This particular Mustang is a 1 owner vehicle with a clean vehicle history report. The car has only 53,341 original miles and runs and drives beautifully. Whether youre looking for a sunny day cruiser, or a sporty daily driver, this Mustang has a lot of life left. You can see this 2007 Ford Mustang GT in greater detail in our Chicago showroom or at www.GatewayClassicCars.com. Please contact 708-444-4488 or chicago@gatewayclassiccars.com. See more at: http://www.gatewayclassiccars.com/CHI/1391/2007-Ford-Mustang-GT
1954 Chevrolet 3800
Ask for: Fort Lauderdale, FL
email: fortlauderdale@gatewayclassiccars.com
For sale in our Fort Lauderdale Showroom is this super cool 1954 Chevrolet 3800 Panel Van. This full ton is ready to be displayed in front of your business; build a food truck out of it or just drive it around town and put many smiles on people faces. There are so many opportunities which make this van so great. The exterior is in very good condition. Beautiful paint on a great and straight body. The advertisement on the van can easily be removed, it is only stickers. You can get an idea of how nice this 3800 would look with your own company logo. The interior is all original and there is room for your creativity. The Chevy is powered by a 6 Cylinder and has plenty of power to move it. The three on the tree shifts through all gears and you will get immediately the classic car driving experience. It is really fun to drive the van. Options are limited with Hub Caps; Seat Belts; Vinyl Interior; Bias Ply Tires but there are no limits for your imagination. Use this Chevy 3800 as a billboard and it will be paid off faster than you would think or buy it as an investment with fun guarantee. There are not many left out there. Now is the time to secure one. After 62 years this classic piece of American History is still all there and ready to go. Wir sprechen deutsch. Let it ride!!! To see 100+ HD pictures and videos of this 1954 Chevrolet 3800, please visit www.GatewayClassicCars.com. For more information or to schedule an appointment to view car in person, please call the Fort Lauderdale showroom at (954) 227-4202 or email us directly at fortlauderdale@GatewayClassicCars.com
Now available in our Dallas/Ft. Worth Showroom is this 2002 4th generation 35th anniversary addition Camaro. The fourth-generation Camaro debuted in 1993 on an updated F-body platform. It retained the same characteristics since its introduction in 1967: a coupé body style with 2+2 seating (with an optional T-top roof) or convertible (reintroduced in 1994), rear-wheel drive, pushrod 6-cylinder and V8 engines. The SS versions (1998-2002) received slightly improved exhaust and intake systems, bigger wheels and tires, a slightly revised suspension for improved handling and grip while retaining ride comfort, an arc-shaped rear wing for downforce, and different gearing ratios for faster acceleration, over the Z28 models. Chevrolet offered a 35th anniversary edition for the 2002 model year and that's exactly what we have here. Motoring this 2002 around is a 5.7-liter LS1 engine controlled by a 6-speed manual. The owner has recently replaced the clutch on this vehicle and replaced the convertible top too. Other options included in this vehicle are: Power seats, Power windows, Power mirrors, Power brakes, Anti-Lock brakes, Power locks, Cd-player/Am/Fm Radio, A/C, Cruise Control, Anti-theft system, & Leather Interior. To view this 2002 Chevrolet Camaro in greater detail, including HD pictures and an HD video, visit www.GatewayClassicCars.com or our Dallas showroom located in DFW Airport, TX. For more information, please call 817-310-9400 or email Dallas@GatewayClassicCars.com
Ask for: HOU
Gateway Classic Cars of Houston is proud to offer this truly unique 2000 Chevrolet Camaro SS convertible with only 24,328 miles showing on its odometer. Underhood is its practically-unused LS1 5.7L V8 engine, sending its power out through its 4-speed 4L60E automatic transmission to its PosiTrac differential with its 3.23:1 axle ratio. This one is finished in Pewter metallic, and has a hand-laid fiberglass hard tonneau cover thats colored to match, which truly gives it its unique sportster look. Inside is its original black cloth interior and GMs Monsoon sound system, complete with remote CD changer and subwoofer. Simply turn the key and listen to the exhaust note and youll wonder what kind of fun the open road will lead to, thanks to a stainless Magnaflow catback exhaust system. Options on this 2000 Chevrolet Camaro SS include: Air Conditioning, AM/FM Radio, 12 Disc CD Changer, Power Convertible Top, Cruise Control, Power Brakes, Power Steering, Power Locks, Power Seats, Power Windows, Anti-Lock Brakes, Tilt Wheel, Traction Control, Keyless Entry, Driver & Passenger Airbags, Anti-Theft System, Cloth Seats, Radial Tires, and Aluminum Alloy Wheels. Its obvious this was a dealers choice in 2000 as the vehicle is fully loaded! This is a car ready to drive across the country, take to a local show, or used as your daily driver.These F-Body Camaros are ever increasing in value. To find one with low miles and in such great condition is getting harder and harder. This is definitely a future classic and a great buy. For more information on this 2000 Chevrolet Camaro SS for sale, please call the Houston showroom at (832) 243-6220; or email us at Houston@GatewayClassicCars.com. To see more of it, go to our website: www.GatewayClassicCars.com, where youll see HD photos and an HD video of it in operation. Alternatively, if you want to see it in person, come to our Houston Showroom, located at 71 Esplanade Blvd, Ste. 100, Houston, TX 77060.
Gateway Classic Cars of Scottsdale is proud to offer this tough and rustic 1978 Ford F150 for sale. This 1978 Ford F150 is a survivor Custom long box truck. This sweet truck still wears its original two-tone green paint that it left the factory with four decades ago. It still presents well but has the nice patina thats so popular today. The new 20 Foose wheels look just right on this classic F150. With a matching green cloth and vinyl interior, youre sure to be comfortable inside as everyone you drive past honks and waves. Powered by Fords reliable 400 CID engine and a C-6 three speed automatic (recently re-sealed), this F150 also has a nice array of options like air conditioning, power steering, power disc/drum brakes, AM/FM/CD radio, and cruise control (currently not working). All of the gauges work and function properly and the steering and front end components have all been replaced in the past two years. This 1978 Ford drives, steers, stops, and handles perfectly. Its an original Arizona truck so it has a rust-free body and frame. These 70s F-series trucks are really gaining momentum in the collector car marketplace. Get this one while its here...which wont be long or before the price on them sky rocket like the Chevy Nova's have! This is a great truck to show off to friends and family. These trucks never disappoint and always show you how reliable a truck can and should be. This is a great truck to own at this price no one will believe how much you spent on this sweet ride. This 1978 Ford F150 for sale includes: AC, AMFM radio, CD player, power brakes, power steering, seat belts, cloth and vinyl interior, radial tires and aluminum alloy wheels. To see 100-plus pictures and a video of this 1978 Ford F150 for sale and for more information or to schedule an appointment to view this vehicle in person, please call the Scottsdale showroom at 623-900-4884 or email us directly at scottsdale@gatewayclassiccars.com
Now for sale in our Houston showroom is this 1980 Chevrolet Corvette. In 1980, the Corvette received an integrated aerodynamic redesign that resulted in a significant reduction in drag. After several years of weight increases, 1980 Corvettes were lighter as engineers trimmed both body and chassis weight all while gas was only $.79 a gallon. This one is in very good condition and shows very well. Looking at the exterior the two-tone paint job is in very good condition that shines in the sun's rays while cruising down the road. The car was repainted several years ago and the darker color is a dark metallic green matched to the silver accent. The stock wheels are in great condition and are wrapped in new BF Goodrich tires. This car has 61271 miles on it but the owner has installed a new crate engine 3,000 miles ago which runs very strong. It is paired to an automatic 3-speed transmission that shifts smoothly throughout the gears & was rebuilt 3k miles ago as well. On the interior, you will find the tan cloth & leather combo that is also in good condition, as the seats, dash, door panels, & carpet are all free from any major tears or cracks. The car has been fitted with a brand new AC system that blows cold! The owner has also installed new brake components (brake lines, master cylinder, & rotors), new T-tops & weather stripping, a new aluminum oversized radiator, & all new tires. The car has been owned within the same family since it was purchased new, the owner also has all service records & maintenance receipts for the past 20 years. Other options on this Corvette include, AC, CD player with USB, T-tops, cruise control, power brakes, power steering, power windows, tilt & telescopic steering wheel, & leather interior. To see all of the HD pictures & HD video on this beautiful Corvette go to www.gatewayclassiccars.com .For additional information on this 1980 Chevrolet Corvette call the Houston Showroom at 832-243-6220 or email houston@gatewayclassiccars.com
1992 Buick Roadmaster
email: dylan@cars1123.com
This awesome low mileage 1992 Buick Roadmaster Estate Wagon is for sale in our Louisville KY showroom. The origins of the Roadmaster name date back to 1936 when Buick added it to their entire line up. Taken out of Buicks line up the namesake would not return until 1991 with the Estate Wagon. Based on the full-size rear wheel drive B platform sales would see a huge increase with almost 10 times as many cars VS the previous year. This 92 is a great example of these classic wagons. The paint is original with the light blue being one of the more desirable colors. The woodgrain running down the side is classic Roadmaster and still shows extremely well. Many say the reason for these being so successful was the V8 engine under that long-rounded hood. This one retains its original L05 V8 which was rated at 180HP and the precursor to the now legendary LT1. The 4l60 transmission shifts out nice and smooth with the OD giving the ability to run 70 on the highway while keeping the RPM��s down. Open the door and a very clean and original blue leather interior compliments the exterior nicely. Have a seat behind the wheel as you begin to sink in it is easy to see why many say these are the most comfortable cars they ever drove. The suspension is soft giving the car that classic floating feel. The highway of course is where these wagons shine and especially when cruising down the open road. The ones in the back seat get the real treat with the moonroof allowing for clear views of the sky above. If you have been thinking about going with a cross-over vehicle but want to be a little different this 1992 Buick Roadmaster Wagon is the perfect fit. Options on this 1992 Buick Roadmaster Estate Wagon include; AC, AM/FM/Cassette, Power Steering, Power Windows, Power Brakes, Power Seat, Seat Belts, Power Locks, Tilt, Seat Belts, Leather Interior, Hub Caps, and Radial Tires. To view this 1992 Buick Roadmaster Estate Wagon, including an HD video, at www.gatewayclassiccars.com. For more information call the showroom at (812) 294 1555 or email louisville@gatewayclassiccars.com.
O'Fallon, IL
Ask for: STL
email: stlouis@gatewayclassiccars.com
For sale in our St. Louis showroom is a 1 owner 1990 Chevrolet Camaro IROC-Z. This IROC is finished in a gloss black exterior paint, which is original,and in great shape. The IROC-Z decals are red, to match the red cloth interior, which is in excellent condition, free many any major wear over the years. Powering this 90' Camaro IROC-Z is a 5.7L 350 CID V8 engine that is backed by a 700R4 automatic transmission. The Posi-Trac rear end with 3.23 gears puts the power to the pavement. The air conditioning has been updated to R-134 freon and blows cold. This being a one owner car, we have copies of the original order sheet, window sticker, and invoice. The odometer reads 41,808, which represents the actual mileage on the car. Options for this 1990 Chevrolet Camaro IROC-Z include: Air Conditioning, AM/FM Radio with Cassette Player, Cruise Control, Power Driver's Seat, Power Windows, Power Locks, Power Steering, Power Brakes, Power Mirrors, Cloth Interior, Seat Belts, Driver Airbag, Tilt Wheel, Posi-Traction, Radial Tires, and Aluminum/Alloy Wheels. The third generation Camaro debuted in the fall of 1981 for the 1982 model year. Nearly every part of the third generation was redesigned. The main focus was to drop weight. Chevrolet succeeded in that goal shedding over 500 pounds from the previous model. The IROC package was introduced in 1985, it was an option available off the standard Z28 package, and it ran until early in the 1990 model year run. IROC or International Race Of Challengers comes from a best of the best style racing series in which top drivers from all walks of racing (NASCAR, Indy, Formula One, etc.) competed against one another for the title of best driver in the world. The IROC-Z package included a number of upgrades: 16 X 8 aluminum/alloy wheels with Z-rated Goodyear Gatorback tires, a lower ride height featuring upgraded front struts, Delco-Bilstein rear shock absorbers and increased spring rates, a Wonderbar or front frame-rail stiffener, and a larger rear stabilizer bar. Cosmetically the IROC-Z received a new grill, body colored ground effects, and the iconic IROC-Z decals on each door. The IROC package included a G92 performance axle as well. This is a great opportunity to own a fairly rare 1990 IROC-Z Camaro, in a beautiful color combination, that has been cared for by its original owner for its entire life. To view this 1990 Chevrolet Camaro IROC-Z in greater detail, including HD pictures and an HD video, visit www.GatewayClassicCars.com or our St. Louis showroom. For more information please call 618-271-3000 or email STL@GatewayClassicCars.com
Gateway Classic Cars of Atlanta is proud to present this 2-owner 1996 Chevrolet Corvette Grand Sport for sale. The RPO Z16 option included the LT4 engine and was only available with the six-speed manual transmission, as this car has. She was also optioned with a uniqueAdmiral Blue w/ White Stripes, and Red Hash Marks paint scheme, that roughly 1,000-units were painted. What makes this LT4 different from the other 5.7L engines in 1996 was simply horsepower and we all know we love horsepower! With factory rated 330 horsepower and 340 ft-lb of torque this 1996 Chevrolet is a blast to drive through all six gears. The Grand Sport came only in Admiral Blue with a white stripe down the middle, 5-spoke black wheels, two red hash marks on the front drivers side fender and special seat trim providing a distinctive look. The Grand Sport also has wider tires and the coupes were outfitted with small rear fender flares. The interior is just as sporty as the outside with black leather sport bucket seats. With only 810 Grand Sport coupes built don't wait and let another one slip through your fingers, just call and grab this one! Options on this 1996 Chevrolet Corvette include: Air Conditioning, AM/FM radio, Cassette, CD player, Targa Top, cruise control, power steering, power brakes, power locks, power 6-position seats, power windows, tilt wheel, anti-lock brakes, traction control, driver and passenger airbags, leather interior, and alloy/aluminum wheels wrapped in radial tires. To view this car in greater detail, including 100+ HD Photos and HD Video, please visit www.gatewayclassiccars.com. For more information, please call the Atlanta showroom at 678-894-4833 or email us at Atlanta@GatewayClassicCars.com.
1969 Mercury Monterey
Ask for: NDY
Gateway Classic Cars is proud to present this 1969 Mercury Monterey. This 69 Mercury is powered by a 390 cubic inch V8 engine and is backed by a 3-speed automatic transmission. Options on this 1969 Mercury Monterey include: Air Conditioning, AM Radio, Power Steering, Power Brakes, Leather Interior, Seat Belts and Bias-ply White Wall Tires wrapped around Steel Wheels with Hub Caps. This 1969 Mercury Monterey is offered for sale with 2,513 actual miles. This is a 3-owner survivor with extremely low original miles. The fuel tank has been replaced recently along with the axle seals. The seller also recently had the carburetor rebuilt. The 7th and final generation Monterey was introduced in 1969 and featured a longer wheelbase (124 inches). These classics have become very popular in recent years in the United States as well as Europe. This classic is as original as they come and still has the plastic wraps on the rear seatbelts. The Monterey is stunning in its original Dark Aqua Metallic exterior with a White top and White leather interior. This classic will show well in the survivor class at the local car shows and is a reliable driver. This 1969 Mercury Monterey for sale is located in our Indianapolis Showroom and can be seen in greater detail, including a High Def video at www.GatewayClassicCars.com. Please call (317)688-1100 or email us at INDY@gatewayclassiccars.com for additional information.
Now for sale in our Houston showroom is this 1966 Ford Mustang in Wimbledon white. This first-generation Mustang debuted with moderate trim changes including a new grille, side ornamentation, wheel covers and gas cap. This 66 needs no introduction and is the most popular and well-known muscle cars of the era. This original "C" code in its original paint (Wimbledon white) that still looks good and is free from any major dents or dings. The trim, bumpers, & emblems are great shape for being 51 years old! This all original car has the original 289 CID V8 engine that is paired to the stock 3-speed "cruise-o-matic" white dot automatic transmission. Looking towards in interior you see the original teal blue vinyl & cloth seats that are in fantastic condition for being original to the car. The dash, carpet, & headliner are also of the same condition. The car came with factory air conditioning and it still works! Blows cold! The car will come with its original owner's manual, original air condition warranty card, registration cards from 1968 & 1969 from the original owner. The car is listed with only 64k original actual miles! The owner has replaced a few key parts on the car including; new shocks on all 4 corners, new carburetor, fuel tank sending unit, heater core, radiator, water pump, & a newer AC compressor. This car was originally $3,339.72 dollars and today you could add this appreciating asset to add to your collection. You won't find many survivor cars out there like this one so make a move on this beautiful 66 Mustang today!! For more information on this 1966 Ford Mustang call the Houston Showroom at 832-243-6220 or email Houston@gatewayclassiccars.com
Gateway Classic Cars is proud to present this 2007 BMW 335i Hardtop Convertible. This 07 BMW is powered by an impressive twin-turbo inline V6 backed by a 6-Speed Automatic Transmission with paddle shifters. This car is rated to produce 320 horsepower and delivered an impressive 0-60 mph time of only 5.4 seconds. Options on this 2007 BMW 335i for sale include: Air Conditioning, AM/FM Radio w/CD player, Convertible Hardtop (Auto), Cruise Control, Heated Seats, Lumbar Support,Power Brakes, Power Steering, Power Locks, Power Seats, Power Windows, Anti-Lock Brakes, Telescopic and Tilt Wheel, Traction Control, Keyless Entry, Ride Control, Seat Belts, Driver Airbag, Passenger Airbag, Anti-Theft System, Leather Interior, and Aluminum/Alloy Wheels wrapped in Bridgestone Driveguard All-Season tires. The paint color on this vehicle is Lemans Blue Metallic and matches perfectly with the beige leather interior. The hardtop on this BMW drops instantly with the push of a button which makes this vehicle a great choice year round. This car provides a great level of comfortability with all the options, yet still has plenty of get up and go for the driving enthusiast. This 2007 BMW 335i for sale is in our Indianapolis Showroom and can be seen in greater detail including an HD video at www.GatewayClassicCars.com. Please call our knowledgeable staff at (317)688-1100 or email us at Indy@GatewayClassicCars.com for additional information.
Ask for: NSH
For sale in our Nashville Tennessee showroom is a classic 1954 Jaguar XK120 Drophead Tribute. This sweet little car is fun and sporty, good looking and eye catching. This 1954 Jaguar XK120 Drophead Tribute for sale is powered by a Volkswagen 1600CC 4 Cylinder that has a low 1,836 miles on it since the build. Being that the power plant has been refreshed you can rest assured when you are at the pump because not you can run regular unleaded and you will not be needing those pesky additives. Filler her up and go. Backing these snappy 4 cylinders is a 4speed manual transmission, grab a gear and carve some corners in this sweet machine. As you cruise though the mountains even the goats in the Alpes will stop munching on there food to grab a glance at you, on lookers at the car shows will stop and stare because let's face it, how many of these have you really seen? The exterior of this 1954 Jaguar XK120 Drophead Tribute for sale is draped in a sexy bright red paint that is best fitted for this fun sports car. The interior of this 1954 Jaguar XK120 Drophead Tribute for sale is rocking dual bucket seats with airplane style lap belts. While you are rolling down the road in your sweet ride you can enjoy the sports car sound of the exhaust system singing that sweet tune of gear grabbing action, watch the tachometer bounce off the yellow line and you are bouncing through the gears. Options on this 1954 Jaguar XK120 Drophead Tribute for sale include: Seats, Fun, Seatbelts, Cool Factor and a fresh set of performance radial tires that are wrapped around custom aluminum alloy wheels. A $237.37 document fee will be added to the sales price. All purchases may be subject to any applicable tax, title, & licensing fees. For more information on this 1954 Jaguar XK120 Drophead Tribute for sale please call the Nashville showroom at 615-213-1800 or visit us on the web at nashville@gatewayclassiccars.com
1984 Chevrolet Monte Carlo
This awesome attitude-infused 1984 Chevrolet Monte Carlo is now available for sale at Gateway Classic Cars in our Milwaukee showroom. From the moment your turn the key in this Indigo Blue muscle classic you feel the power churning from the 480 horsepower 355 small block V8 with an aggressive note being sung to you via a combination of a high lift camshaft, Hooker long tube headers, and 3 mandrel bent exhaust coming out to a pair of Flowmaster mufflers. Despite having a small block built for racing being under the hood of this car, the engagement of the transmission hence the streetable feel of this car is kept in check by a 3,000 RPM stall. This makes the car feel like it can be street driven to work any day of the week or have the thunderous feel of going to the track; owners choice. The specs of the drivetrain include: Chevy bowtie heads bolted to a #010 casted 4 bolt main block featuring 2.02 intake valves and 1.60 exhaust valves, both made of stainless steel, with 67cc combustion chambers. Also has Crower 4340 steel connecting rods holding JE Racing ceramic coated flat top pistons. Opening these valves at the center of the engine is a high lift mechanical flat tappet camshaft featuring 255/263 duration, 106 degree lobe separation and .585 lift, connected to the crankshaft via a double row timing chain. Camshaft is lubricated with a high-volume oil pump being fed by an 8-quart Moroso oil pump. This engine is neatly dressed with a chrome air cleaner as well as a pair of tall valve covers with dual breather covers as well as an Edelbrock RPM Performer intake manifold with a Holley double pump 750cfm carburetor sitting handsomely up top neighboring an Accel distributor and ignition coil. This beautiful Monte also features a new Kenwood car stereo deck and new speakers all the way around. Options on this fast and furious machine include; power windows, power brakes, power steering, seat belts, a/c, heat, and performance tires. This 1984 Monte Carlo is for sale in our Milwaukee Showroom and can be viewed in greater detail including 100+ HD pictures and HD video at www.GatwayCLassicCars.com. For additional information, please call our knowledgeable staff at (262) 891-4253 or email us at Milwaukee@gatewayclassiccars.com. Hours: Monday through Saturday, 9 AM to 5 PM CST. If you would like to see it in person, come to our Milwaukee Showroom, located at 9949 58th Place Suite 400, Kenosha WI 53144
1951 Chevrolet Styleline
Now for sale in our Houston Showroom is one awesome 1951 Chevrolet Styleline Deluxe. This is one fine automobile, and has some very tasteful upgrades. For starters, this 51 Styleline has been enhanced with a 12 volt electrical system, front disc brakes, electric wipers, a new exhaust with a new dual-tip muffler, and a new gas tank. This car went through a complete reconditioning approximately 8 years ago, including the drivetrain, with both the engine and transmission receiving rebuilds; and more recently, the rear end was reconditioned just about a year ago. With only about 2500 total miles on the drivetrain since being redone, this car is ready for some sedate, stylish cruising. Powered by its torquey 216.5 CID Inline 6 cylinder, backed by its three on the tree column-shifted manual transmission, sending the twist to a set of 3.55:1 rear gears, youll find yourself gliding down the road getting plenty of thumbs ups from your fellow travelers. Inside, youll find a custom-installed Vintage Air system housed in a custom center console, giving it a real factory look. The Vintage AC installation was done very tastefully underhood as well, with everything tucked away neatly and routed cleanly. In that same center console, one will find a modern audio system, complete with AM/FM/CD Player, and cupholders. The gray-green and tan interior is finished in soft velour seating surfaces, and trimmed with gray synthetic leather that matches the velours gray, on both the console and the seat frames. The headliner is in superb shape, and color matches the tan velour on the door panels and seats. This is one retro-cool interior, built for comfort and stylish cruising. The exterior is as nice as the interior, bathed in a dark gray metallic paint that has a touch of green added to it, which just gleams in the sunlight. All of the chrome is in great condition, and its custom 18 chromed aluminum wheels are wrapped in Riken Raptor ZR rated tires, really filling the wheel wells out nicely, and adding to its cruiser look. Options on this 1951 Chevrolet Styleline Deluxe for sale include Air Conditioning, Custom Chrome-plated Aluminum Wheels, Electric Wipers, Performance Radial Tires, and AM/FM/CD Player radio. For more information on this tight 1951 Chevrolet Styleline Deluxe, contact our Houston showroom at (832) 243-6220; or email us at Houston@GatewayClassicCars.com. To see more of it, go to our website: www.GatewayClassicCars.com, where youll see HD photos and an HD video of it in operation. Alternatively, if you want to see it in person, come to our Houston Showroom, located at 71 Esplanade Blvd, Ste. 100, Houston, TX 77060
Thank you for your interest in this awesome 1977 Chevrolet 1500 presented by Gateway Classic Cars of Tampa! This excellent truck is one mean hot rod with a built up 383 CID Stoker and a Drag Racing rear-end and is ready to tear it up at the strip!! This truck went through a complete frame-off resto-modification 10 years ago and is still in showroom and drag strip condition! The 383 Stroker was built and installed by Boyd Racing along with the 700R4 transmission. Bolted to this power block is a set of long-tube headers, Edelbrock intake manifold, Holley carburetor, and an MSD ignition system and she sounds MEAN! The rear end is a tough and reliable Ford 9 with 2 super-wide Hoosier 31x18.50R150LT 99H Tires providing you with all the traction and grip you need to take others off the line! The interior is in excellent condition finished in both black and blue cloth and vinyl leather. All the gauges work with individual toggles for the fan, ignition and fuel pump. The exterior paint is in pristine condition with virtually no flaws, and the bow tie pint strip along the side of the truck gives it the right amount of class it needs! Shes turn-key and ready to go and fires up on the first crank on a cold start! To see 100+ HD pictures and videos of this custom 1977 Chevrolet 1500, please visit GatewayClassicCars.com. For more information or to schedule an appointment to view car in person, please call the Tampa showroom at (813)645-6200 or email us directly at Tampa@GatewayClassicCars.com. See more at: http://www.gatewayclassiccars.com/TPA/915/1977-Chevrolet-1500


text:
Q: Como calcular a distância entre dois pontos e calcular distância ABC, CAB, BAC? em C criei em sala de aula esse programa que calcula a distância entre dois pontos e em seguida dá a opção de 3 caminhos diferentes: ABC, BAC ou CAB.
Gostaria de dicas para otimizar meu código, ideias de como melhorá-lo e também dicas de novas funcionalidades...
segue o código:
    #include <stdio.h>
    #include <stdlib.h>
    #include <math.h>

    int main(int argc, char *argv[])
    {
        float ax, bx,cx, ay, by, cy;
        float AB, BC, AC;
        float distancia;
        int rota;

        printf("Digite o valor de Ax\n");
        scanf("%f", &ax);
        printf("Digite o valor de Ay\n");
        scanf("%f", &ay);
        printf("Digite o valor de Bx\n");
        scanf("%f", &bx);
        printf("Digite o valor de By\n");
        scanf("%f", &by);
        printf("Digite o valor de Cx\n");
        scanf("%f", &cx);
        printf("Digite o valor de Cy\n");
        scanf("%f", &cy);

        AB=sqrt(pow((bx-ax),2)+pow((by-ay),2));
        printf("A distancia entre os pontos A e B eh %f\n", AB);

        AC=sqrt(pow((cx-ax),2)+pow((cy-ay),2));
        printf("A distancia entre os pontos A e C eh %f\n", AC);
        BC=sqrt(pow((cx-bx),2)+pow((cy-by),2));
        printf("A distancia entre os pontos B e C eh %f\n\n", BC);
        printf("Escolha uma rota: ABC = 1, BAC = 2 ou CAB = 3\n");
        scanf("%d", &rota);
        switch (rota)
        {
          case 1:
            distancia = AB + BC;
            printf("A distancia entre os pontos ABC eh %f\n", distancia);
          break;

          case 2:
            distancia = AB + BC;
            printf("A distancia entre os pontos BAC eh %f\n", distancia);
          break;

          case 3:
            distancia = AB + BC;
            printf("A distancia entre os pontos CAB eh %f\n", distancia);
          break;

          default :
        printf("ROTA INVALIDA!!!");

        }

        system("pause");
        return 0;
    }

A: A dica que eu dou, então, é sobre estilo.
Você já tem alguma noção de estrutura de dados? Seria interessante deixar este código mais limpo, usando structs e encapsulando algumas ações repetidas em sub-rotinas.
Por exemplo, os pontos podem ser abstraídos dessa forma:
typedef struct point{
  float xcoord;
  float ycoord;
} point;

/* Uso: point A = set_point(4.0,3.0) */
point set_point(float xcoord, float ycoord){
  point ret;

  ret.xcoord = xcoord;
  ret.ycoord = ycoord;

  return ret;
}

Com isto, a distância entre dois pontos pode ser calculada por esta função:
float distance(point A, point B){
  float dx = A.xcoord - B.xcoord;
  float dy = A.ycoord - B.ycoord;

  return sqrt(dx*dx + dy*dy);
}

Não recomendaria usar a função pow, dado que a multiplicação nativa é(?) mais rápida.
Outra coisa seria essa frase "A distância entre os pontos ABC é".... Esse fraseado é confuso. Que tal "O comprimento da rota A-B-C é"...?
Eu também sugeriria algo mais verboso na parte da coleta dos valores das coordenadas dos pontos, algo como "digite a coordenada x do ponto A", "digite a coordenada y do ponto C".

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