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.

Toxicological aspects of topical silver pharmaceuticals.

Silver is generally considered to present a relatively low toxic threat to humans because unintentional exposure to large doses of the noble metal is quite rare. However, as the intentional utilization of silver in pharmaceutical preparations and devices increases, subtle toxic effects of silver may be predictable and expected. The present review examines the scientific literature, primarily covering the past 10 years, dealing with reports describing various types of silver toxicity. These reports consist of both in vitro and in vivo data dealing with immunological, mesenchymal, neural, and parenchymal cell types. Particular emphasis is given to (1) the use of silver in topical antimicrobial preparations as toxicity relates to absorption through dermal wounds into the systemic circulation and possible effects on delayed wound healing, (2) possible local silver toxicity via iontophoretic devices, (3) current theories relating to the toxicological mechanism of action of silver.

Effect of nordihydroguaiaretic acid and ibuprofen on bleomycin and hyperoxia-induced changes in lung superoxide dismutase, prostaglandins and lethality.

Mortality in hamsters injected intratracheally with bleomycin (bleo) (0.25 units) and exposed to 70% oxygen is significantly increased over 14 days when they receive nordihydroguaiaretic acid (NDGA, 10 mg/kg per day S.C.). Mortality in bleo +O2 challenged hamsters treated with ibuprofen (10 mg/kg per day) subcutaneously was not significantly increased. The increased mortality in NDGA treated animals is not the result of augmented superoxide formation, since no change in lung superoxide dismutase occurred. Among the prostaglandins measured, NDGA produced the greatest elevation in 6-keto-PGF1 alpha (stable product of prostacyclin) and thromboxane B2 (stable product of thromboxane A2).

Effect of cadmium on lung lysosomal enzymes in vitro.

Labilization of lysosomal enzymes is often associated with the general process of inflammation. The present study investigated the effect of the pneumotoxin cadmium on the release and activity of two lung lysosomal enzymes. Incubation of rat lung lysosomes with cadmium resulted in the release of beta-glucuronidase but not acid phosphatase. The failure to "release" acid phosphatase appears to be the result of a direct inhibitory effect of cadmium on this enzyme. The K1 for cadmium was determined to be 26.3 microM. The differential effect of cadmium on these two lysosomal enzymes suggests that caution should be exercised in selecting the appropriate enzyme marker for assessing lysosomal fragility in the presence of this toxicant. Furthermore, the differential basal release rate of the two enzymes from lung lysosomes may reflect the cellular heterogeneity of the lung.

Effect of antibody to transforming growth factor beta on bleomycin induced accumulation of lung collagen in mice.

BACKGROUND: Increased production of transforming growth factor beta (TGF-beta) seems to have an important role in the pathophysiology of bleomycin induced lung fibrosis. This is attributed to the ability of TGF-beta to stimulate infiltration of inflammatory cells and promote synthesis of connective tissue, leading to collagen deposition. METHODS: The study was designed to evaluate the antifibrotic potential of TGF-beta antibody in mice treated with bleomycin, which is a model of lung fibrosis. Under methoxyflurane anaesthesia, each mouse received intratracheally either 50 microliters sterile isotonic saline or 0.125 units bleomycin in 50 microliters. Within five minutes after the instillation, mice received into the tail vein 100 microliters non-immune rabbit IgG, TGF-beta 2 antibody, or a combination of TGF-beta 2 and TGF-beta 1 antibodies at various dose regimens. Mice were killed 14 days after the instillation and their lungs processed for morphological and biochemical studies. RESULTS: Administration of 250 micrograms of TGF-beta 2 antibody after instillation of bleomycin followed by 100 micrograms on day 5 and 100 micrograms on day 9 significantly reduced the bleomycin induced increases in the accumulation of lung collagen from 445.8 (42.3) micrograms/lung to 336.7 (56.6) micrograms/lung at 14 days. Similarly, the combined treatment with 250 micrograms TGF-beta 2 antibody and 250 micrograms TGF-beta 1 antibody after bleomycin instillation followed by 100 micrograms of each antibody on day 5 also caused a significant reduction in bleomycin induced increases in lung collagen accumulation and myeloperoxidase activity at 14 days. CONCLUSIONS: These results suggest that TGF-beta has an important role in the aetiology of bleomycin induced lung fibrosis; the neutralisation of TGF-beta by systemic treatment with its antibodies offers a new mode of pharmacological intervention which may be useful in treating lung fibrosis.

Attenuation of amiodarone-induced lung fibrosis and phospholipidosis in hamsters by taurine and/or niacin treatment.

Therapeutic use of amiodarone (AD), an effective antiarrhythmic drug, is associated with serious pulmonary toxicity (e.g., fibrosis and phospholipidosis). In the present study, we tested if taurine and/or niacin, which prevent bleomycin-induced lung toxicity, could prevent AD-induced lung toxicity in hamsters. AD alone significantly increased lung hydroxyproline (an index of fibrosis) and lung phospholipid (an index of phospholipidosis) levels to 154 and 133% of their control counterparts at 21 days, respectively. However, treatment of hamsters with taurine, niacin or taurine + niacin for 6 days before AD, and daily thereafter, significantly decreased subsequent AD-induced collagen accumulation. Similarly, phospholipid levels in niacin + AD and taurine + niacin + AD groups were decreased significantly compared to AD alone. We conclude that taurine and niacin administered p.o. either singly or in combination can significantly decrease AD-induced increases in lung collagen deposition and phospholipidosis and may, therefore, be potentially useful in reducing AD-induced pulmonary toxicity.

Effects of thiourea on pulmonary vascular permeability and on lung and plasma histamine levels in rats.

The intraperitoneal administration of thiourea (TU) to mature male rats results in a significant increase in lung vascular permeability to Evans Blue dye (EBD). On the other hand, young, sexually immature rats are resistant to this effect. The increase in lung vascular permeability in response to TU in mature rats is associated with corresponding increases in lung and plasma histamine levels. The correlation of increases in lung and plasma histamine in response to TU is similar to that reported for ammonium salts which produce similar pulmonary edema.

Effects of dietary alteration on trimethylaminuria as measured by mass spectrometry.

The urine of a 9-year-old boy suffering from extreme body odour was assayed for trimethylamine (TMA) by gas chromatography-mass spectrometry. A trapping column bed, specifically designed for volatile compounds, was used in the mass spectrometry. The diagnosis of rare trimethylaminuria was confirmed by the presence of appreciable urinary TMA. Urinary TMA concentrations were reduced to virtually nil after the elimination of the major choline sources (fish and eggs) from the diet.

Effects of thiourea tolerance on plasma histamine, and lung vascular permeability.

Adult male rats treated with a lethal edematogenic dose of thiourea (TU) (10.0 mg/kg, intraperitoneally) responded with significant elevations in plasma histamine, lung vascular permeability and 100% mortality over a subsequent 24-h period. When rats were pretreated with a small non-lethal dose of TU (0.5 mg/kg) and subsequently challenged with the lethal dose at 1, 4, 8, 16 and 32 days later, there was complete protection against death for at least 8 days and partial protection for an additional 24 days. This decrease in mortality correlated quite closely with reduced plasma histamine levels and diminished pulmonary vascular permeability. The results suggest that reduced exposure of the pulmonary vasculature to histamine may offer a partial explanation for tolerance to thiocarbamide compounds in the rat.

The effect of bleomycin on the uptake and incorporation of [14C]choline into phospholipids in hamster lung tissue slices.

Intratracheal administration of the anticancer drug bleomycin to hamsters produced an increase in the uptake and incorporation of [14C]choline into phospholipids of lung slices in vitro. The stimulatory effect is opposite to the results obtained previously using [14C]acetate and would appear to occur distal to cytidine diphosphocholine. Although alternate explanations are possible, the results are consistent with morphological evidence, published by others, indicating an increase in lung phospholipid following bleomycin treatment, and illustrate the significance of precursor selection when evaluating the effects of xenobiotics on phospholipid synthesis.

Pulmonary toxicity of inhaled and intravenous talc.

Talc (magnesium silicate) is a widely used, generally considered benign substance. It is principally used as an inert filler material in drug tablets or as a drying ingredient in baby powders. However, in both cases inappropriate use can lead to severe pulmonary toxicological responses. On the one hand, intravenous injection of 'solubilized', CNS active pills can produce microemboli in small pulmonary vessels. This can lead to various degrees of granuloma formation, compromised pulmonary function, or death. Overzealous application of baby powder can also produce severe pulmonary complications if the infant inspires the powder. Although the data are relatively scarce, a number of reports suggest the existence of a chronic problem in this area.

Non-enzymatic covalent binding of radioactivity from [14C]thiourea to rat lung protein.

In vitro covalent binding of radioactivity from [14C]thiourea (TU) to rat lung protein occurs in the presence of 100% CO2. Prior boiling of lung slices results in a subsequent increase in covalent binding. Covalently bound radioactivity was released from rat lung homogenates and acid-insoluble protein obtained from animals treated with [14C]TU. The release was achieved with ammonium ion. One possibility suggested by these results is that the bound species may involve binding of isothiocyanic acid via a thiocarbamylation reaction.

Interaction of thiourea with rat lung protein.

Administration of the pulmonary edemagenic agent [14C]Thiourea [( 14C]TU) to adult rats results in heterogeneous binding to lung protein. Indirect evidence suggests that TU may be binding to the amino acid cysteine, possibly via the SH moiety. The nature of the adduct is not known, but appears to involve one major and several minor species.

Effects of two isomers of hexachlorocyclohexane (HCH) on cortical beta-adrenoceptors in rat brain.

Two isomers of hexachlorocyclohexane (HCH) have profound effects on the acquisition of kindled seizures. The gamma-isomer (lindane, gamma-HCH) increases the rate of acquisition in a dose-related manner whereas the beta-isomer (beta-HCH) has the opposite effect, retarding the rate in a dose-related manner. There is evidence that adrenergic influences may play a role in seizure generalization and in this study we examined the effect of these two isomers of HCH on the binding of a ligand to beta-adrenoceptors. Tritiated dihydroalprenolol was used as the ligand in an assay using cortical tissue from rats after 10 days of treatment with 10 mg/kg, p.o. of isomers or corn oil. Results showed a statistically significant decrease in the number of beta-adrenoceptors in cortical tissue taken from gamma-HCH-treated rats. In contrast, there was a statistically significant increase in the number of beta-adrenoceptors cortical tissue taken from beta-HCH-treated rats. Neither treatment produced a significant change in KD. We concluded that the beta-adrenoceptor system in rat cortex is differentially affected by the isomers of HCH. Because the kindling model of epilepsy is sensitive to modulations in adrenergic function, this system may be involved in the pro- and anticonvulsant effects of these HCH isomers on kindling acquisition.

Effect of bilirubin on the spectrophotometric and radionuclide assay for serum angiotensin-converting enzyme.

The effect of bilirubin on serum angiotensin-converting enzyme (ACE) activity was studied with spectrophotometric and radionuclide assays. In the spectrophotometric assay addition of bilirubin to normal serum from dog, mouse, and human produced a dose-related inhibition of ACE activity. A 50% decrease in human ACE activity was produced by the addition of approximately 250 mg/L in vitro. Serum from icteric patients with elevated bilirubin was also associated with a reduction in ACE activity in the spectrophotometric assay. A 50% decrease in ACE activity in these samples was associated with a serum bilirubin of approximately 220 mg/L. In the radionuclide assay, however, addition of bilirubin to normal human serum failed to reduce measured ACE activity. The use of a radionuclide assay for serum ACE in clinical samples offers the advantage of less interference from serum bilirubin.

SAR of the effect of endotoxin on serum angiotensin converting enzyme in the mouse.

Administration of intact lipopolysaccharide from E. coli and S. minnesota to mice produced a lowering of serum angiotensin converting enzyme (ACE). However, when either lipid A or carbohydrate-free endotoxin from S. minnesota-Re595 was administered, a rise in serum ACE occurred. A possible explanation for these respective effects may be related to an indirect effect on lung perfusion, on the one hand, or a direct toxic effect to endothelial cell membranes, on the other.

The value of serial serum angiotensin converting enzyme determinations in hospitalized patients with lung disease.

Abnormal serum angiotensin converting enzyme (ACE) activity has been reported in various human lung disorders and in laboratory animals with acute lung injuries. To test the value of serum ACE activity as an indicator of lung damage and its assistance in diagnosis or prognosis, 328 serum samples were obtained from 108 hospitalized patients with lung disease and 26 normal subjects. When patients were clinically grouped by disease entity, only the sarcoidosis group showed elevated mean serum ACE. Significantly increased serum ACE was found in 17 patients with various lung diseases (15% of hospitalized patients) 12 of whom also had concomitant liver disease. It is hypothesized that the liver may play a role in the normal metabolism of ACE being released by lung endothelial injury. Significantly low levels were seen in many acute and chronic lung injuries; specifically the groups with chronic obstructive lung disease, lung cancer, acute pneumonia, aspiration pneumonitis, gram-negative sepsis, acute myocardial infarction, and congestive heart failure. Serial measures of ACE in 71 patients with lung injuries showed that significantly decreasing levels over successive days were associated with a very high mortality. A single ACE measurement did not predict the presence or extent of lung injury, or aid in diagnosis or prognosis, but serial levels are of value prognostically.

Potentiation of paraquat lethality in mice by bacterial lipopolysaccharide pretreatment.

Mice treated with Escherichia coli endotoxin (500 micrograms/kg i.p.) 24 and 1 hr before paraquat dichloride (50 mg/kg i.p.) had a 2-fold increase in 7-day cumulative mortality compared to those injected with buffered saline before paraquat. The duration of the prior exposure time to endotoxin appears to be an important determinant of the potentiating effect as more mice died after 24 than 1 hr pretreatment. The potentiating effect of endotoxin on paraquat-induced lethality is not due to increased uptake of the toxicant. Lung values of radioactivity from [14C]paraquat were not significantly different between endotoxin and buffered saline-treated mice. Despite the potentiating effect of endotoxin on paraquat-induced lethality, it appears that the lipopolysaccharide is able to provide some protection to the pulmonary capillary endothelium because pretreatment reverses an increase in serum angiotensin converting enzyme. Although not addressed in the present investigation, possible mechanisms for the potentiating effect of endotoxin on paraquat-induced lethality may involve superoxide anion generation, superoxide dismutase inhibition or both.