Suppression of bleomycin-induced nitric oxide production in mice by taurine and niacin.

The effects of taurine (T) and niacin (N) on the influx of inflammatory cells and nitric oxide (NO) levels in bronchoalveolar lavage fluid (BALF) and expression of inducible NO synthase (iNOS) mRNA and iNOS protein in lungs were evaluated in the bleomycin (BL)-mouse model of lung fibrosis. Mice were placed into four groups: saline-instilled (SA) with a control diet (CD) (SA + CD); saline-instilled with TN (1% taurine in water + 2.5% (w/w) niacin in diet) (SA + TN); BL-instilled with CD (BL + CD); and BL-instilled with TN treatment (BL + TN). There was no difference in differential cell counts in BALF between the SA + CD and SA + TN control groups. Intratracheal instillation (IT) of BL (0.1 U/mouse) in mice stimulated an early influx of neutrophils followed by an increase in lymphocytes and macrophages in the BL + CD group. Taurine and niacin treatment significantly reduced the numbers of neutrophils, lymphocytes, and macrophages in the BL + TN group and caused significant reductions in BL-induced increases in the lung hydroxyproline content at 14 and 21 days in the BL + TN group. The mice in the SA + CD and SA + TN control groups had low levels of NO in BALF, whereas mice in the BL + CD group as compared to the SA + CD control group had elevated levels of NO from day 3 through day 21. Taurine and niacin treatment caused significant reductions in BL-induced increases in NO levels in BALF from mice in the BL + TN group at 7, 14, and 21 days as compared to the corresponding BL + CD group. The increases in NO levels in BALF from the BL + CD group were associated with elevated levels of iNOS gene expression and protein in the lungs. RT-PCR analysis of total RNA isolated from the lungs indicated that taurine and niacin treatment suppressed the BL-induced increases in iNOS message and iNOS protein. The ability of taurine and niacin to suppress the BL-induced increased production of NO secondary to decreases in iNOS mRNA and protein appears to be one of the mechanisms for their anti-inflammatory and antifibrotic effects.

Taurine and niacin block lung injury and fibrosis by down-regulating bleomycin-induced activation of transcription nuclear factor-kappaB in mice.

The effects of taurine (T) and niacin (N) on bleomycin (BL)-induced increased production of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1alpha, IL-6, and transforming growth factor-beta (TGF-beta) levels in the bronchoalveolar lavage fluid (BALF), and increased collagen content and nuclear factor-kappaB (NF-kappaB) activation in the lungs were investigated in mice. The mice were intratracheally instilled with saline (SA) or BL (0.1 U/mouse/50 microliter) under ketamine and xylazine anesthesia. They had ad libitum access to diet containing 2.5% niacin (w/w) or the same control diet (CD) and water with and without taurine (1%) 3 days before intratracheal instillation and throughout the study. The mice were sacrificed at different times for collecting BALF and lungs, which were appropriately processed for various measurements. Treatment with taurine and niacin attenuated the BL-induced increases in proinflammatory cytokines such as IL-1alpha, TNF-alpha, IL-6, and TGF-beta in BALF and lung hydroxyproline content of the mice in BL + TN groups. Reverse transcription-polymerase chain reaction analysis of total RNA from whole lung was performed to assess the induction of TNF-alpha and IL-1 mRNAs as markers of NF-kappaB activation. The NF-kappaB DNA-binding activity in whole-lung extract was evaluated by electrophoretic mobility shift assay. This revealed a progressive increase in NF-kappaB activation and IkBalpha depletion in lungs from mice in BL + CD groups from day 1 through day 21 compared with the corresponding SA + CD control groups. Treatment with taurine and niacin generally inhibited the BL-induced increases in the nuclear localization of NF-kappaB and preserved IkappaBalpha protein in BL + TN groups. This may be one of the mechanisms for the antifibrotic effect of taurine and niacin.

Effects of pirfenidone on transforming growth factor-beta gene expression at the transcriptional level in bleomycin hamster model of lung fibrosis.

This study was undertaken to investigate whether treatment with the antifibrotic drug pirfenidone (PD) down-regulates the bleomycin (BL)-induced overexpression of transforming growth factor (TGF)-beta gene in the lungs. Hamsters were intratracheally instilled with SA or BL (6.5 U/kg/4 ml) under anesthesia. They were fed a diet containing 0.5% PD or the same control diet (CD) without the drug 2 days before and throughout the study. After the animals were sacrificed, their lungs were appropriately processed. The BL treatment elevated the total influx of inflammatory cells, including macrophages, by severalfold at different days in bronchoalveolar lavage fluid (BALF) from hamsters in BL + CD groups, relative to the corresponding SA + CD control groups. Treatment with PD significantly (P

Effects of pirfenidone on procollagen gene expression at the transcriptional level in bleomycin hamster model of lung fibrosis.

A time course study was carried out to elucidate the mechanisms for antifibrotic effect of pirfenidone (PD). Hamsters were intratracheally (i.t.) instilled with saline (SA) or bleomycin (BL) (7.5 units/kg/5 ml). The animals were fed a diet containing 0.5% PD or the same control diet (CD) without the drug 2 days before and throughout the study. The animals were sacrificed at various times after instillation. The lung hydroxyproline level in BL + CD groups was gradually increased and peaked at 21 days to 181% of the SA + CD control. The BL + PD-treated groups showed a gradual decrease in their lung collagen content, showing a maximum reduction of 40% at day 21. The lung malondialdehyde levels of the BL + CD groups were increased by several-fold of the corresponding SA + CD groups at various times. The lung prolyl hydroxylase (PH) activities in the BL + CD groups were also increased by several-fold of the corresponding SA + CD groups at these time points. The hamsters in the BL + PD showed a gradual decrease in the lung malondialdehyde levels from 10 to 21days compared with their corresponding BL + CD groups. Treatment with PD also reduced the lung PH activities in the BL + PD groups compared with the corresponding BL + CD groups. However, PD failed to manifest any direct inhibitory effect on PH activity in vitro. BL treatment increased the lung procollagen I and III gene expressions in the BL + CD groups by several-fold at varying times compared with the corresponding SA + CD, and treatment with PD in the BL + PD groups significantly down-regulated the BL-induced overexpression of these genes. Studies evaluating the regulation of these genes at the transcriptional level revealed PD significantly reduced the transcription of PC I at 14 days. Our results indicate that the antifibrotic effect of PD was partly due to suppression of the BL-induced inflammatory events and partly due to down-regulation of BL-induced overexpression of lung procollagen I and III genes.

Pirfenidone inhibits PDGF isoforms in bleomycin hamster model of lung fibrosis at the translational level.

Pirfenidone (PD) is known for its antifibrotic effects in the bleomycin (BL) hamster model of lung fibrosis. We evaluated whether pretreatment of hamsters with PD could influence the effects of BL-induced overexpression of platelet-derived growth factor (PDGF)-A and PDGF-B genes and proteins in the same model of lung fibrosis. We demonstrate elevated levels of PDGF-A and PDGF-B mRNAs in bronchoalveolar lavage (BAL) cells from lungs of BL-treated compared with saline control hamsters by RT-PCR analysis. However, these levels were not altered in BAL cells obtained from BL-treated hamsters on diets containing 0.5% PD. Western blot analysis of BAL fluid for PDGF isoforms demonstrated that PD treatment inhibited the synthesis of both PDGF-A and PDGF-B isoforms. PD treatment also decreased the mitogenic activity in the BAL fluid from BL-treated hamster lungs. Taken together, these data provide evidence that the protective effects of PD against BL-induced lung fibrosis may be mediated by a reduction in PDGF isoforms produced by lung macrophages.

Regulation of transforming growth factor-beta1 mRNA expression by taurine and niacin in the bleomycin hamster model of lung fibrosis.

We have reported that taurine (T) and niacin (N) inhibit the expression of procollagen type I and type III genes at the level of gene transcription in the bleomycin (BL) hamster model of lung fibrosis. In the present study, we have investigated the effects of TN in diet on the temporal expression of transforming growth factor-beta1 (TGF-beta1) mRNA and TGF-beta1 protein production in the same model of lung fibrosis to determine whether the decreased transcription of procollagen genes is associated with downregulation of TGF-beta1 mRNA. Our results demonstrate that expression of TGF-beta1 mRNA in lungs is increased in BL-treated hamsters in the BL + control diet (CD) group, compared to saline controls in the saline-instilled (SA) + CD group, by 3.5-, 2.5-, 4-, and 2-fold at 3, 7, 14, and 21 d, respectively, and TN treatment caused significant decreases in TGF-beta1 mRNA expression in BL-treated animals in the BL + TN group from Day 3 through Day 21. In addition, TN treatment also reduced TGF-beta1 protein in bronchoalveolar lavage fluid (BALF) from BL-treated animals in the BL + TN group. These decreases in TGF-beta1 mRNA and TGF-beta1 protein correlated with decreased lung collagen content in hamsters in the BL + TN group as demonstrated in our earlier study. To confirm that the TGF-beta1 activity observed in BALF is reflected at the transcriptional level, total RNA was isolated from lavaged cells. Reverse transcriptase-polymerase chain reaction analysis demonstrated maximal expression of TGF-beta1 mRNA transcripts in BL-treated lavaged cells from animals in the BL + CD group and only low levels were detected in both saline control groups, and in BL + TN-treated lavaged cells. Nuclear runoff analysis indicated that TN-mediated reduction of TGF-beta1 mRNA steady-state levels was a result of decreased gene transcription, suggesting a transcriptional downregulation mechanism. Our results indicate that the combined treatment with TN ameliorates BL-induced lung fibrosis, at least in part, via inhibition of TGF-beta1 mRNA expression.

Procollagen gene expression is down-regulated by taurine and niacin at the transcriptional level in the bleomycin hamster model of lung fibrosis.

Taurine (T) and niacin (N) have previously been found to block the accumulation of collagen in the bleomycin (BL) model of interstitial pulmonary fibrosis. The present study was designed to evaluate whether the mechanism for the antifibrotic effect of combined treatment with taurine and niacin involves the down-regulation of BL-induced overexpression of procollagen I and III messenger ribonucleic acid (mRNA) levels in lungs. Hamsters were intratracheally instilled with three consecutive doses of saline or BL at weekly intervals (2.5, 2.0, 1.5 units/5 ml/kg). Four groups of animals were fed a diet throughout the experiment containing either 2.5% taurine and 2.5% niacin or the same diet without the drugs. The four groups were saline-instilled with the control diet (SA + CD), saline-instilled with TN in the diet (SA + TN), BL-instilled with the control diet (BL + CD), and BL-instilled with the TN diet (BL + TN). Steady state transcript levels in total RNA prepared from lungs of all four groups were determined at 0, 3, 7, 14 and 21 days after the last BL instillation by slot blot and Northern blot analyses. Results indicate that procollagen I mRNA levels are elevated compared with saline control by 2.5-, 2.4- and 2.0-fold at 7, 14, and 21 days after the last dose of BL instillation, respectively. Dietary treatment with taurine and niacin decreased the steady state level of BL-induced increases of procollagen I mRNA from day 0 through 21. We observed a similar pattern of procollagen III inhibition by taurine and niacin from day 3 through day 21. Transcription of procollagen I and III genes was readily detected in nuclei prepared from BL-treated lung samples at 14 days after treatment. In contrast, transcription of procollagen I and III genes was barely detectable in nuclei prepared at the same time point from BL + TN treated lungs. Our results suggest that procollagen I and III gene expression in BL-induced lung fibrosis in hamsters is transcriptionally down-regulated by combined treatment with taurine and niacin.

Molecular mechanisms of antifibrotic effect of interferon gamma in bleomycin-mouse model of lung fibrosis: downregulation of TGF-beta and procollagen I and III gene expression.

The present study was undertaken to elucidate the mechanism for the antifibrotic effect of interferon gamma (IFN-gamma) in the bleomycin (BL)-mouse model of lung fibrosis. The expression of transforming growth factor (TGF-beta) and procollagen I and III and their mRNAs was investigated in the BL-mouse model of lung fibrosis with and without IFN-gamma treatment by Northern and slot blot analyses. Temporal changes in the content of procollagen and TGF-beta mRNAs in the lungs of mice receiving saline or BL by intratracheal route, with and without IFN-gamma treatment by intramuscular route, were quantitated. The level of TGF-beta mRNA increased rapidly and peaked at day 5, whereas the levels of mRNAs for procollagens alpha 1(I) and alpha 1(III) peaked at 10 days after BL instillation. The peak levels of these mRNAs in BL-treated animals were five- to sevenfold higher than those of the control. The increase in TGF-beta mRNA in the lungs of BL-treated mice preceded the increase in the synthesis of type I and type III procollagen mRNAs. BL treatment also increased the hydroxyproline content significantly from 3 to 14 days as compared to the corresponding saline control groups. A maximal increase to 447 micrograms/lung from 223 micrograms/lung in saline control was obtained at 10 days after instillation. Daily treatment with IFN-gamma markedly reduced the BL-induced increases in the mRNA levels of TGF-beta, and procollagen alpha 1(I) and alpha 1(III) without any effect on the lung level of beta-actin mRNA. IFN-gamma treatment also caused significant reduction in the BL-induced increase in the lung hydroxyproline content from 417 to 283 micrograms/lung at 7 days and from 447 to 264 micrograms/lung at 10 days. It may be concluded from the findings of the present study that the cellular mechanisms for the antifibrotic effect of IFN-gamma in the BL-mouse model of lung fibrosis are to initially downregulate the BL-induced overexpression of TGF-beta mRNA, and subsequently procollagen mRNAs, leading to a decreased collagen content.

Isolation of phenol-degrading Bacillus stearothermophilus and partial characterization of the phenol hydroxylase.

Bacillus stearothermophilus BR219, isolated from river sediment, degraded phenol at levels to 15 mM at a rate of 0.85 mumol/h (4 x 10(6) cells). The solubilized phenol hydroxylase was NADH dependent, exhibited a 55 degrees C temperature optimum for activity, and was not inhibited by 0.5 mM phenol.

Degradation of gallic acid by Aspergillus flavus.

Aspergillus flavus utilized gallic acid as sole carbon source and increased in presence of glucose and sucrose. The enzymes were inducible in nature and during degradation of gallic acid. 4-carboxy, 2-hydroxy cis, cis-muconic acid and pyruvic acid were detected. The estradiol (meta) fission of gallic acid by A. flavus was postulated.