Dietary intake of pirfenidone ameliorates bleomycin-induced lung fibrosis in hamsters.

There are no clinically efficacious drugs available for preventing the development of pulmonary fibrosis (PF). In the present study, we tested the antifibrotic potential of pirfenidone (PD) in the bleomycin (BL) hamster model of PF. Hamsters were intratracheally instilled with isotonic saline solution or BL (7.5 U/kg/5 ml). The animals were fed control diet containing 0.5% PD or the same diet without the drug 2 days before and throughout the study. The four groups were as follows: saline-instilled and fed the control diet (SCD); saline-instilled and fed the same diet containing PD (SPD); BL-instilled and fed the control diet (BCD); and BL-instilled and fed the same diet containing PD (BPD). The animals were killed at 21 days after intratracheal instillation and their lungs processed for various assays. The lung hydroxyproline levels, an index of PF, in SCD, SPD, BCD, and BPD groups were 949, 970, 1759, and 990 micrograms/lung, respectively. The SOD activity and malondialdehyde equivalent levels in the corresponding groups were 443, 524, 612, and 499 units/lung and 56, 49, 108, and 63 nmol/lung, respectively. The lung prolyl hydroxylase activities in the SPD, BCD, and BPD groups were 87%, 147%, and 93% of the control (SCD) group (4.2 x 10(4) dpm/lung/30 minutes), respectively. The lung myeloperoxidase activities were 97%, 236%, and 159% of the control group (0.73 units/lung), respectively. BL alone caused significant increases in all the biochemical markers of lung toxicity, and dietary intake of PD minimized the BL toxicity as reflected by significant decreases in all the above markers.(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary supplementation with taurine and niacin prevents the increase in lung collagen cross-links in the multidose bleomycin hamster model of pulmonary fibrosis.

Taurine and niacin have been previously found to block the accumulation of collagen in lung in the multidose bleomycin hamster model of pulmonary fibrosis. Previous studies have found an increase in the pulmonary collagen cross-links dihydroxylysinonoroleucine (DHLNL) and hydroxypyridinium (OHP) in the single dose bleomycin rat model. In this study, we asked if taurine and niacin would block the increase in DHLNL and OHP in the multidose bleomycin hamster model of lung fibrosis. Hamsters were intratracheally instilled with three consecutive doses of saline or bleomycin sulfate 1 week apart (2.5, 2.0, 1.5 units/5 mL/kg). Animals were fed diet containing either 2.5% niacin and 2.5% taurine or control diet throughout the experiment. The four groups were saline-instilled with control diet (SCD), bleomycin instilled with control diet (BCD), bleomycin-instilled with taurine-niacin in diet (BTN), and saline-instilled with taurine-niacin in diet (STN). Animals were sacrificed at 1, 4, and 8 weeks after the last bleomycin instillation. Hydroxyproline per lung in the BCD group was significantly elevated by 38, 56, and 60% over the SCD group at 1, 4, and 8 weeks, respectively. There were no statistically significant differences among the four groups in DHLNL (mmole) per mole collagen at the 1 or 8 week time point. At four weeks, DHLNL was significantly elevated by 46.4% in the BCD group over the SCD group. The OHP (mmole) per mole of collagen at 1 and 4 weeks in the BCD group was not statistically different from the SCD group.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural and biochemical changes in lungs of 3-methylindole-treated rats.

Effects of a single dose of 3-methylindole (3-MI) (250 mg/kg intraperitoneally) were studied at different times ranging from 12 hours to 2 weeks post-treatment (PT). Microscopic study revealed mild Clara cell injury 24 hours PT and mucus hyperplasia 24 hours to 2 weeks PT. Diffuse type I alveolar epithelial cell necrosis occurred at 48 hours, followed by type II cell hyperplasia. Septal edema and accumulation of interstitial and capillary polymorphonuclear leukocytes and perivascular mixed mononuclear inflammatory cells accompanied the injury and repair. A gradual resolution of lesions with persistent mononuclear inflammatory cellular clusters at septal junctions, focal septal fibrosis, and accumulation of alveolar macrophages was evident at 1 and 2 weeks PT. Collagen, measured as hydroxyproline, in 3-MI-treated rats was significantly increased to 130% and 139% of control (3.0 mg/lung) at 1 and 2 weeks PT, respectively. Biphasic peaks of plasma 6-keto-prostaglandin F1 alpha occurred at 12 to 24 hours and at 96 hours PT with 3-MI and thromboxane B2 was elevated 12, 48, and 96 hours PT. Right ventricular/left ventricular and septal weight was increased to 120% and 140% of the control 1 and 2 weeks PT. We concluded that 3-MI induces alveolar septal injury in the rat with relatively complete repair of the alveolar epithelium and residual mild focal septal fibrosis and pulmonary hypertension 2 weeks PT. Arachidonic acid-derived mediators and inflammation are associated with 3-MI-induced lung injury.

Etiology of sarcoplasmic reticulum calcium release channel lesions in doxorubicin-induced cardiomyopathy.

Alterations in the native function of the ryanodine-sensitive Ca2+ release channel complex of sarcoplasmic reticulum (SR) isolated from rat cardiac ventricles during acute and chronic exposure to doxorubicin are examined. Compared to control SR, actively loaded SR from animals exposed to a single intravenous dose of doxorubicin exhibit faster rates of doxorubicin-induced Ca2+ release and the occupancy of [3H]ryanodine is significantly enhanced with subsequent exposure of SR membranes to doxorubicin in vitro. One week after acute exposure to doxorubicin in vitro, the EC50 for activation of the binding of [3H]ryanodine by Ca2+ is not significantly different from control SR. However, the persistence of doxorubicin-sensitized SR channels appears to be latent since repeated exposure to doxorubicin in vitro significantly enhances receptor occupancy in SR obtained from the treated rats compared to control SR. Ryanodine receptors from rats chronically exposed to doxorubicin consistently exhibit a higher sensitivity to activation Ca2+ which persists at least 4 weeks following the last injection of drug. Chronic exposure produces a concomitant reduction in the capacity of [3H]ryanodine binding sites. The marked decrease in receptor density observed with SR from doxorubicin-treated rats coincides with significant reduction in body weight, suggesting a possible influence of nutrition. However, sodium dodecyl sulfate polyacrylamide electrophoresis indicates no significant loss of the high molecular weight subunit of the ryanodine receptor, suggesting that loss of [3H]ryanodine-binding capacity may be the result of progressive and permanent channel desensitization. Consistent with desensitized receptors, membrane vesicles prepared from rats chronically exposed to doxorubicin take up significantly more Ca2+ and exhibit significantly reduced rates of doxorubicin or Ca2+/ryanodine induced Ca2+ release. The data demonstrates (i) doxorubicin inflicts cumulative SR channel lesions in vivo, (ii) a persistent sensitization of the SR channel to activation by Ca2+ and (iii) a significant and apparently irreversible reduction in the number of functional channel complexes.

Anthraquinone-sensitized Ca2+ release channel from rat cardiac sarcoplasmic reticulum: possible receptor-mediated mechanism of doxorubicin cardiomyopathy.

Rat cardiac membrane vesicles enriched in biochemical markers of the junctional region of sarcoplasmic reticulum (SR) and exhibiting ruthenium red-sensitive rapid Ca2+ release have been prepared. Doxorubicin and seven congeners are shown to enhance the binding of [3H]ryanodine to the ryanodine receptor with a strong structural requirement. Doxorubicin enhances the binding of [3H]ryanodine to SR membranes and soluble receptor preparations and induces Ca2+ release from SR vesicles in a highly Ca2(+)-dependent manner, suggesting that anthraquinones promote the open state of the junctional Ca2+ release channel by increasing the affinity of the Ca2+ activator site for Ca2+. Doxorubicin reduces the Kd of [3H]ryanodine binding solely by enhancing the rat of association. Caffeine competes for the same site with anthraquinones, because the caffeine-activated binding of [3H]ryanodine is inhibited by doxorubicin and vice versa. The acute effect of doxorubicin on the cardiac Ca2+ release channel is fully reversible; however, long term treatment (up to 24 hr) with doxorubicin increases the sensitivity of the preparation to subsequent acute challenge with doxorubicin. The thiol-reductive agent dithiothreitol enhances, whereas the reactive disulfide 4,4'-dithiodipyridine reduces, the doxorubicin-enhanced binding of [3H]ryanodine. These results demonstrate that the acute and chronic cardiotoxicity of anthraquinones may be accounted for by a receptor-mediated mechanism. Our findings suggest that the chronic effects observed with the clinical use of anthraquinones may be the result of a receptor-mediated shift in the redox equilibrium of allosteric thiols at the ryanodine receptor complex, which in turn leads to long term sensitization of the Ca2+ release channel.

Effects of three cysteine pro-drugs on bleomycin-induced lung fibrosis in hamsters.

The cysteine pro-drug Z2196 ((2RS, 4R)-2-methylthiazolidine carboxylic acid) and two drugs with methyl esters attached to Z2196 (Z2197 and Z2199) were evaluated for antifibrotic effects in the hamster bleomycin model of lung fibrosis. Each drug or phosphate-buffered saline (PBS) was given daily (300 mg/kg intraperitoneally) for 2 days before intratracheal instillation of bleomycin (7.5 units/kg) or saline for an additional 13 days. Lung collagen measured as hydroxyproline was significantly increased to 138% of the control groups in the PBS + Bleomycin treated group, but the Z2196 + Bleomycin group was increased to 108% and was not statistically different from controls. Protein content of bronchoalveolar lavage supernatant in PBS + Bleomycin treated hamsters was significantly increased to 326% of controls. The protein content of bronchoalveolar lavage supernatant for all cysteine pro-drug + Bleomycin treated hamsters was increased to 160% of PBS + Bleomycin treated hamsters. All the Bleomycin treated hamsters had significantly more cells and more neutrophils recovered in bronchoalveolar lavage than controls. The PBS + Bleomycin treated hamsters had significantly more lymphocytes in bronchoalveolar lavage than all the other treatment groups. The Z2196 + Bleomycin and Z2197 + Bleomycin hamsters had significantly less monocytes in BALF than PBS + Bleomycin hamsters. The lung total sulfhydryl and nonprotein sulfhydryl in PBS + Bleomycin treated hamsters were increased to 210% and 253% of controls, respectively, whereas in Z2196 + Bleomycin hamsters they were increased to 152% and 153%, respectively. Histopathology of PBS + Bleomycin hamsters showed a diffuse mixed mononuclear alveolitis, multifocal fibrosis and peribronchiolar fibrosis, whereas Z2196 + Bleomycin hamsters showed notably less alveolitis and fibrosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Early over-expression of low-affinity [3H]ryanodine receptor sites in heavy sarcoplasmic reticulum fraction from dystrophic chicken pectoralis major.

Heavy sarcoplasmic reticulum (SR) membranes enriched in [3H]ryanodine receptor have been isolated from pectoralis major (PM) of normal line 412 and dystrophic line 413 chickens paired at various stages during post-hatch development. Normal PM 2 days ex ovo yields 17% lower protein recovery in the heavy SR subfractions compared to preparations from paired dystrophic PM (0.80 vs. 0.96 mg/g PM, respectively). By 2 weeks ex ovo, protein recovery in normal SR subfractions decreases over 3-fold to 0.24 mg/g PM, whereas yields from dystrophic PM increase to 1.67 mg/g PM. Dystrophic preparations consistently give 7-9-fold higher recoveries of protein in heavy SR subfractions compared to normal PM when examined at 2, 4, and 5.5 weeks ex ovo. [3H]Ryanodine binding to normal SR from PM 2 days ex ovo exhibits nonlinear Scatchard plots which resolve into high-(Kd,app = 18 nM; Bmax = 1.7 pmol/mg protein) and low-(Kd,app = 532 nM; Bmax = 2.6 pmol/mg protein) affinity binding sites, whereas dystrophic preparations exhibit only high-affinity [3H]ryanodine binding sites (Kd,app = 31 nM; Bmax = 2.1 pmol/mg protein). Both normal and dystrophic PM have similar capacities to bind [3H]ryanodine (2.6 vs. 2.0 pmol/g PM, respectively) at 2 days ex ovo. However, at 2, 4, and 5.5 weeks ex ovo the density of high-affinity [3H]ryanodine binding sites in normal SR drops dramatically to 3.5, 1.2, and 0.4 pmol/mg protein, respectively, and corresponds with disappearance of the low-affinity binding sites. In marked contrast, heavy SR membranes from dystrophic PM 2, 4, and 5.5 weeks ex ovo, maintain their high-affinity binding sites for [3H]ryanodine and exhibit high densities of low-affinity binding sites (Kd,app = 725-4500 nM; Bmax = 15.4-25.1 pmol/mg protein). Early developmental over-expression of [3H]ryanodine binding capacity in dystrophic PM ranges from 34-fold to 388-fold that of normal PM at 2 weeks and 5.5 weeks, respectively, and correspond to the intensity with which high molecular weight doublets of Mr 340,000 and 320,000 stain on SDS-PAGE. Low-affinity [3H]ryanodine binding sites of dystrophic SR exhibit 4-6-fold higher sensitivity to activation by Ca2+ and altered sensitivity to activation by caffeine and adenine nucleotides. These results demonstrate that over-expression of junctional SR and [3H]ryanodine receptors having altered radioligand binding properties is a very early event in the post-hatch development of dystrophic PM.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of repeated administration of N-acetyl-L-cysteine on sulfhydryl levels of different tissues and bleomycin-induced lung fibrosis in hamsters.

N-Acetyl-L-cysteine (NAC), 50, 100, 200, or 400 mg/kg, was injected intraperitoneally once a day for 13 days. No change was seen in the total sulfhydryl (TSH) and nonprotein sulfhydryl (NPSH) contents of the liver, kidney, and plasma at any dose. The heart TSH level remained unchanged, but the NPSH level was increased from the control value of 16 nmol/mg to 18, 19, and 18 nmol/mg protein at 50, 100, and 200 mg/kg, respectively. The lung TSH and NPSH levels both were increased from the control values of 65 and 8 nmol/mg to 80 and 16 nmol/mg protein, respectively, at 200 mg/kg. The lung TSH level at 400 mg/kg NAC was not changed, but the NPSH level increased to 13.5 nmol/mg protein. The ratio of TSH to NPSH levels in the liver and kidney was 4:1, whereas in the lung and heart it was 7:1 and 8:1, respectively. Based on amount per milligram of protein, TSH and NPSH levels were highest in the liver, followed by the amounts in the kidney, heart, and lung. The lung had the lowest level of TSH and NPSH. The daily treatment with NAC (200 mg/kg) for 13 days after and 2 days before intratracheal injection of bleomycin (7.5 U/kg) had little effect on lung collagen accumulation. The lung collagen level measured as hydroxyproline in bleomycin and in NAC plus bleomycin was significantly increased to 175% and 183% of the control levels, respectively. There was no difference in the lung hydroxyproline content between the control and NAC groups. The histopathology study also revealed no marked difference between the bleomycin and bleomycin plus NAC groups. Alternatively, treatment with NAC (200 mg/kg) for 13 days before bleomycin made the animals more susceptible to bleomycin toxicity and tended to add to the bleomycin-induced accumulation of collagen in the lung. NAC per se caused no mortality at any dose. The lung TSH and NPSH levels in bleomycin-treated (7.5 U/kg) hamsters were increased to 136% and 111% of control, respectively, whereas the TSH and NPSH levels both were increased to 155% of the levels of their respective controls in hamsters in the NAC plus bleomycin group. The differential effects of NAC treatment on the sulfhydryl content of tissues, the treatment's inability to alter the course of bleomycin-induced lung inflammation and collagen accumulation, and the potential for exacerbation of lung toxicity in response to repeated administration of NAC before exposure to fibrogenic agents are discussed.

Effects of bleomycin on 14C-proline uptake, its incorporation into proteins and hydroxylation in collagenous proteins of hamster lung slices.

The uptake of 14C-proline by lung slices and its incorporation into trichloroacetic-acid (TCA)-insoluble proteins and hydroxylation in collagenous proteins were studied. They were found to be linearly increased with the incubation time for as long as 8 h. The effects of different concentrations of bleomycin on these parameters were subsequently investigated. Bleomycin at 10(-3) M significantly reduced the uptake of 14C-proline to 75% of the control value. The incorporation of 14C-proline into TCA-insoluble proteins was reduced to 84%, 78% and 75% of the control at 2.5 X 10(-4) M, 5 X 10(-4) M and 10(-3) M concentrations of bleomycin, respectively. Bleomycin failed to alter the proline pool size of the lung slices at any of these concentrations. In addition, bleomycin also inhibited the hydroxylation of 14C-proline into collagen to 72% and 69% of the control at 5 X 10(-4) M and 10(-3) M, respectively. These findings show that bleomycin-induced increases in collagen synthesis in vivo is probably secondary to inflammatory reactions of the lung and bleomycin per se has little effect on increased collagen synthesis responsible for lung fibrosis.

Role of prostaglandins in pathogenesis of bovine mastitis induced by Escherichia coli endotoxin.

Four doses (5 to 100 micrograms, 1 dose/quarter) of Escherichia coli endotoxin were introduced into lactating mammary glands of 2 cows. There was no effect on milk prostaglandin (PG) E2 concentration, except that the concentration was increased from 200 pg/ml of milk to 1,060 pg/ml at post-treatment hour (PTH) 8 in cow 1 and from 75 to 420 pg/ml at PTH 4 in cow 2 after the highest dose 100 micrograms. Endotoxin caused a dose-dependent increase in milk PGF2 alpha concentrations in both cows. After the highest dose, PGF2 alpha was maximally increased from 200 to 3,500 pg/ml at PTH 4 in cow 1 and from 250 to 2,000 pg/ml in cow 2 at PTH 8. The instillation of 50 micrograms of endotoxin in all 8 quarters of 2 more lactating cows caused no significant (P greater than 0.05) changes in milk PGE2 and thromboxane B2 concentrations, whereas milk PGF2 alpha was significantly increased from the base-line value of 642 to 2,683, 1,189, and 2,281 pg/ml at PTH 4, 8, and 12, respectively. The 6-keto-PGF1 alpha was also significantly increased from the base-line value of 305 to 871, 631, and 600 pg/ml at the corresponding times, respectively. A marked increase in vascular permeability, as judged by high concentrations of serum albumin in the whey, was observed as early as PTH 4 and peaked at PTH 12 followed by a gradual decline, although it remained significantly increased over the control for 48 hours after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of intratracheal administration of bleomycin on GSH-shuttle enzymes, catalase, lipid peroxidation, and collagen content in the lungs of hamsters.

Intratracheal administration of one unit of bleomycin increased significantly the glucose-6-phosphate dehydrogenase activity to 147, 135, 163, 207, 278, and 148% of the control at 1, 2, 4, 7, 14, and 21 days post-treatment, respectively. The glutathione peroxidase activity was significantly increased to 147 and 124% of the control at 7 and 14 days post-treatment, respectively. The glutathione reductase and catalase activities were significantly elevated to 132, 174, and 145% of the control in the former and 160, 213, and 160% of the control in the latter case at 7, 14, and 21 days post-treatment, respectively. The amount of thiobarbituric acid reacting products as an index of lipid peroxidation was found to be significantly increased to 251, 248, and 253% of the control at 4, 7, and 14 days after treatment, respectively. The amount of total lung collagen in treated hamsters was significantly increased to 140, 197, and 245% of the control at 7, 14, and 21 days post-treatment, respectively. These findings indicate that the increases in the activities of antioxidant enzymes in lungs of bleomycin-treated hamsters result from the multiple cyto-dynamic events such as infiltration of inflammatory cells, and reparative and proliferative processes which generally follow lung injury caused by any oxidant.

Effects of paraquat on plasma glucose, cortisol, catecholamines, and insulin in the beagle.

Paraquat is known to produce hyperglycemia in rodents, although the mechanisms responsible for the hyperglycemic effect of paraquat are not clearly understood. In this study we report the effects of paraquat (25 mg/kg iv) on plasma glucose, cortisol, catecholamines, and insulin in dogs. Paraquat caused a significant increase in plasma glucose level at all times of measurement except at 0.5 hr following paraquat administration. The cortisol levels after paraquat treatment were increased to 133, 305, 279, 418, 517, 672, and 567% above zero time control at 0.5, 1.5, 3, 6, 12, and 24 hr, and at death time respectively. The catecholamines at these times were increased to 42, 17, 72, 118, 138, 226, and 435% above the control. In contrast to cortisol and catecholamines, the plasma insulin levels after paraquat treatment were significantly decreased at 3, 6, 12, and 24 hr. These hormonal observations can explain, at least in part, the observed hyperglycemia, since all promote hyperglycemic states. For example, cortisol is gluconeogenic, catecholamines are glycogenolytic, and lower insulin levels result in depressed peripheral glucose utilization.

The effects of paraquat and superoxide dismutase on pulmonary vascular permeability and edema in mice.

Intraperitoneal administration of 50 mg/kg paraquat dichloride to mice significantly increased pulmonary vascular permeability at 24 and 48 hr, as measured by 125I-albumin content of alveolar lavage. Lung edema, measured by lung weight as percent body weight, was significantly increased 48 hr after paraquat treatment. Intravenous administration of four doses of superoxidase dismutase at 12-hr intervals (i.e., one before and three after paraquat treatment) failed to inhibit paraquat-induced increased pulmonary vascular permeability and pulmonary edema. Superoxide dismutase treatment also failed to reduce mortality and had no significant effect on the death time course in animals challenged with paraquat. The results of this study suggest that acute toxic effects of paraquat, such as increased pulmonary vascular permeability and pulmonary edema, may not be mediated through the generation of superoxide anion.