Perturbation of copper (Cu) homeostasis and expression of Cu-binding proteins in cadmium-resistant lung fibroblasts.

To probe mechanisms of cadmium (Cd) damage to the lung extracellular matrix (ECM), we developed Cd-resistant (CdR) rat lung fibroblasts (RFL6) by incubation with graded concentrations of Cd. CdR cells downregulated lysyl oxidase (LO), a copper (Cu)-dependent enzyme essential for crosslinking of collagen and elastin in the ECM, in conjunction with upregulation of other Cu-binding proteins including Cu,Zn-superoxide dismutase (SOD1), copper chaperone for SOD1 (CCS1), metallothionein (MT), and Menkes P-type ATPase (ATP7A), a Cu transporter in the membrane of the Golgi apparatus, as well as gamma-glutamylcysteine synthetase (gamma-GCS), an enzyme for glutathione biosynthesis. Reduction and loss of cytoplasmic distribution of LO in CdR cells were accompanied by its dislocation with the Menkes P-type ATPase and the endoplasmic reticulum marker. CdR cells displayed a defect in LO catalytic activity but an enhancement in Cu,Zn-SOD catalytic activity consistent with the protein expression levels of these enzymes. Although long-term Cd exposure of cells enhanced the Menkes P-type ATPase protein expression, actually, it reduced Cu-dependent catalytic activity of this enzyme in parallel with the deficiency of LO. The low level of 64Cu bound to the LO fraction and the high level of 64Cu bound to the MT fraction provide direct evidence for limitation of Cu bioavailability for LO existing in the CdR cells. These results suggest that downregulation of LO is linked with upregulation of other Cu-binding proteins and with alteration in Cu homeostasis in the CdR phenotype.     

Downregulation of lysyl oxidase and upregulation of cellular thiols in rat fetal lung fibroblasts treated with cigarette smoke condensate.

Lysyl oxidase (LO), a copper-dependent enzyme, plays a critical role in the formation and repair of the extracellular matrix (ECM) by catalyzing the crosslinking of elastin and collagen. To better understand mechanisms of cigarette smoke (CS)-induced emphysema, we examined changes in LO and its substrates, i.e., elastin and collagen type I, the major components of cellular thiols, i.e., metallothionein (MT) and glutathione (GSH), and gamma-glutamylcysteine synthetase (gamma-GCS), a key enzyme for GSH biosynthesis, in cigarette smoke condensate (CSC)-treated rat fetal lung fibroblasts (RFL6). Exposure of RFL6 cells to CSC decreased levels of LO catalytic activity, mRNA, and protein, i.e., the 46 kDa preproenzyme, the 50 kDa proenzyme and the 32 kDa mature enzyme in a dose-dependent manner. In addition, CSC also inhibited the expression of collagen type I and elastin, substrates of LO and important components of the lung ECM. Meanwhile, cellular thiols including MT and GSH as well as gamma-GCS were markedly upregulated in CSC-treated cells. To evaluate modulation of LO expression by cellular thiols, we further examined the effect of increased levels of GSH on LO expression at protein and catalytic levels. Interestingly, exposure of cells to glutathione monoethyl ester, a GSH delivery system, effectively elevated cellular GSH levels and induced a dose-dependent decrease in levels of the protein species and catalytic activity of LO. These results suggest that upregulation by CSC of cellular thiols may play an important role in the downregulation of LO and subsequently destabilization of the lung ECM in CS-induced emphysema.     

The critical role of the cellular thiol homeostasis in cadmium perturbation of the lung extracellular matrix

Cadmium (Cd) inhalation can result in emphysema. Cd exposure of rat lung fibroblasts (RFL6) enhanced levels of metal scavenging thiols, e.g., metallothionein (MT) and glutathione (GSH), and the heavy chain of γ-glutamylcysteine synthetase (γ-GCS), a key enzyme for GSH biosynthesis, concomitant with downregulation of lysyl oxidase (LO), a copper-dependent enzyme for crosslinking collagen and elastin in the extracellular matrix (ECM). Cd downregulation of LO in treated cells was closely accompanied by suppression of synthesis of collagen, a major structure component of the lung ECM. Using rats intratracheally instilled with cadmium chloride (30 μg, once a week) as an animal model, we further demonstrated that although 2-week Cd instillation induced a non-significant change in the lung LO activity and collagen synthesis, 4- and 6-week Cd instillation resulted in a steady decrease in the lung LO and collagen expression. The lung MT and total GSH levels were both upregulated upon the long-term Cd exposure. Emphysematous lesions were generated in lungs of 6-week Cd-dosed rats. Increases of cellular thiols by transfection of cells with MT-II expression vectors or treatment of cells with GSH monoethyl ester, a GSH delivery system, markedly inhibited LO mRNA levels and catalytic activities in the cell model. Thus, Cd upregulation of cellular thiols may be a critical cellular event facilitating downregulation of LO, a potential mechanism for Cd-induced emphysema.     

Bleomycin induces upregulation of lysyl oxidase in cultured human fetal lung fibroblasts

Aim:

To investigate the mechanism of bleomycin (BLM)-induced pulmonary fibrosis.

Methods:

Cultured human fetal lung fibroblast (HLF) cells were exposed to bleomycin (BLM) at 0–30 μg/mL for 24 h. Western blot analysis was used to detect lysyl oxidase (LO) protein expression. Real-time RT-PCR was used to detect LO mRNA level. LO catalytic activity was measured using diaminopentane as a substrate and Amplex red as a hydrogen peroxide probe. Copper (Cu) concentration was detected by flame atomic absorption spectrophotometry.

Results:

Exposure of HLF cells to BLM at 10 μg/mL and 30 μg/mL increased LO catalytic activity to 130% and 158% of the control in the conditioned media. The expression of LO mRNA was increased to 5.5-fold of the control in HLF cells exposure to BLM at 3 μg/mL. BLM at 3 μg/mL also increased the expression of 46 kDa preproLO, 50 kDa proLO and 32 kDa mature LO to 219%, 130%, and 135% of the control, respectively. The Cu concentrations in conditioned media of cultured HLF cells exposed to BLM (10 and 30 μg/mL) were increased significantly to 1.48 and 2.46-fold of the control, respectively.

Conclusion:

Bleomycin induces upregulation of LO in cultured human fetal lung fibroblasts, which may be the mechanism of bleomycin-induced pulmonary fibrosis.     

Cadmium-induced alterations in RNA metabolism in cultures of Chinese hamster cells sensitive to and resistant to the cytotoxic effects of cadmium.

A variant population (CdR) of cultured Chinese hamster cells (line CHO) was derived that is more than 100 times as resistant to the cytotoxic effects of Cd2+ than is the parent population. The effects on RNA metabolism of exposure to sublethal concentrations of Cd2+ were studied in CHO and CdR. Exposure to 2 X 10(-7) M CdCl2 for 24 h resulted in increased polysome content (1.2 times) and increased uridine or adenosine incorporation into heterogeneous nuclear RNA (1.2-1.4 times) and messenger RNA ((1.5-1.7 times) in both populations. Measurement of ATP pool specific activity following exposure to radiolabeled adenosine showed that increased incorporation reflects increased synthesis. The equivalence of CHO and CdR in dose-response in terms of stimulated RNA synthesis and their disparity in dose-response in terms of cytotoxic effects indicate that the systems involved in conferring protection against the lethal effects of Cd2+ are not similarly involved in attenuating the effects on RNA metabolism.     

Protease-antiprotease imbalance in the pathogensis of emphysema and chronic bronchial injury: A potential target for drug development

The hypothesis that emphysema might be caused by an imbalance between proteases and antiproteases in the lungs arose shortly after the discovery of the premature and familial occurrence of emphysema in persons with homozygous alpha-1-protease inhibitor deficiency. Experimental evidence for support of this hypothesis has come from experimental induction of emphysema in animals by proteases. The ability of proteases to induce emphysema is proportional to their elastolytic potency. The importance of elastin degradation as a cause of the development of emphysema has also come from experiments of genetic models in which elastin crosslinking is prevented. The neutrophilis believed to be the most likely source of elastase in the induction of the emphysema of smokers. Alpha-1-protease inhibitor is the most important antiprotease. Alpha-1-antiprotease is susceptible to oxidative inactivation by cigarette smoke or by endogenous oxidants. Oxidants from cigarette smoke may also interfere with proper elastin repair. Serine proteases have also been shown to produce secretory cell metaplasia in the central intrapulmonary bronchi of hamsters. The discovery of secretory leukoprotease inhibitor in sputum and its production by bronchial secretory cells raises the possibility that secretory cell metaplasia in humans may also be caused by an imbalance between proteases and antiproteases. Although evidence for the validity of the proteaselantiprotease hypothesis of the phogenesis of emphysema and chronic bronchitis in human smokers is still indirect, the development of supplemental antiprotease therapy seems rational for the prevention of progression of these diseases in persons who are unable to stop smoking.     

Interaction between cadmium and copper in relation to the collagen metabolism of embryonic chick bone in tissue culture

To investigate the interaction between Cd and Cu in relation to the content of collagen in bone, femurs obtained from 9-day-old chick embryos were cultivated with a combination of Cd and Cu concentrations of 8.9 microM and below for 4 days. When 2.23 or 4.45 microM Cd was added to the medium containing 4.45 or 8.90 microM Cu, the presence of both Cd and Cu caused a remarkable decrease in collagen synthesis compared with a decrease in collagen synthesis caused by either Cd or Cu alone. The results show that Cd and Cu caused an interactive inhibition of collagen synthesis, which was not due to an increase in collagen degradation by Cd and Cu. At concentrations showing inhibition, Cd caused an increase in Cu content and Cu caused an increase in Cd content. The increase in Cd content was mainly caused by the accumulation of Cd in bone mineral. The increase in Cu content was due to binding to metallothionein-like protein in the cytosol induced by Cd. The relationship between inhibition and the increase in both Cd and Cu was not confirmed after division of the bone into diaphysis and epiphysis. The present study showed that Cu aggravated the bone matrix damage caused by Cd.     

Oral exposure to cadmium and mercury alone and in combination causes damage to the lung tissue of Sprague-Dawley rats

Environmental presence and human exposure to heavy metals in air and cigarette smoke has led to a worldwide increase in respiratory disease. The effects of oral exposure to heavy metals in liver and kidney structure and function have been widely investigated and the respiratory system as a target is often overlooked. The aim of the study was to investigate the possible structural changes in the lung tissue of Sprague-Dawley rats after oral exposure for 28 days to cadmium (Cd) and mercury (Hg), alone and in combination at 1000 times the World Health Organization’s limit for each metal in drinking water. Following exposure, the general morphology of the bronchiole and lungs as well as collagen and elastin distribution was evaluated using histological techniques and transmission electron microscopy. In the lungs, structural changes to the alveoli included collapsed alveolar spaces, presence of inflammatory cells and thickening of the alveolar walls. In addition, exposure to Cd and Hg caused degeneration of the alveolar structures resulting in confluent alveoli. Changes in bronchiole morphology included an increase in smooth muscle mass with luminal epithelium degeneration, detachment and aggregation. Prominent bronchiole-associated lymphoid tissue was present in the group exposed to Cd and Hg. Ultrastructural examination confirmed the presence of fibrosis where in the Cd exposed group, collagen fibrils arrangement was dense, while in the Hg exposed group, additional prominent elastin was present. This study identified the lungs as target of heavy metals toxicity following oral exposure resulting in cellular damage, inflammation and fibrosis and increased risk of respiratory disease where Hg showed the greatest fibrotic effect, which was further, aggravated in combination with Cd.     

The effects of cadmium, copper or zinc on formation of embryonic chick bone in tissue culture

Femurs from 9-day-old chick embryo were cultivated for 6 days by the roller-tube method in the presence of Cd, Cu or Zn. Cd (5.0 μM and above) and Cu (2.5 μM aand above) caused a decrease in collagen content of both diaphysis and epiphysis, mainly due to inhibition of collagen synthesis. In addition, Cd and Cu each showed a tendency to inhibit an increase in Ca content of diaphysis, where intraperiosteal ossification could be observed. Alkaline phosphatase (ALP) activity was decreased by Cd (5.0 μM and above) or Cu (10 μM and above) in diaphysis. On the other hand, Zn at 50 μM and above inhibited an increase in Ca content of the diaphysis with a remarkable elevation of ALP activity in the medium. At this time, Zn did not decrease the collagen content of the diaphysis so strongly.

Histological observations revealed that Cd and Cu each decreased both calcified and uncalcified osteoid tissue at 2.5 μM, while Zn at 100 μM decreased calcified tissue but increased uncalcified osteoid tissue. As Zn accumulated particularly in diaphysis and deposited at the edge of calcified tissue, it was suggested that Zn inhibited calcification physicochemically. It was concluded that Cd or Cu would induce bone damage represented by osteoporosis, whereas Zn would induce osteomalacia.     

Mechanism of noradrenaline-induced heterologous desensitization of adenylate cyclase stimulation in rat heart muscle cells: increase in the level of inhibitory G-protein alpha-subunits

The mechanism of heterologous desensitization of adenylate cyclase stimulation was studied in cultured neonatal rat heart muscle cells. After culturing of the cells for 3 days in the presence of 1 microM noradrenaline there was in addition to a 52% decrease in isoproterenol-stimulated adenylate cyclase activity, a lessening of the stimulation of beta-adrenoceptor-independent adenylate cyclase by guanosine-5'-O-(thiotriphosphate) and forskolin by 24 and 34%, respectively. The decrease in receptor-independent adenylate cyclase stimulation by forskolin, but not the attenuation of isoproterenol-stimulated adenylate cyclase activity, was abolished by pertussis toxin (PTX) pretreatment of the cells. Gi, the inhibitory G-protein of adenylate cyclase was therefore quantitated. Labelling of the Mr approximately 40 kDa PTX substrates in membranes of noradrenaline-treated cells was increased by 70% as shown by pertussis toxin-catalyzed ADP ribosylation of heart cell membranes. This increase was also seen in the presence of an excess of purified beta gamma-subunits of transducin and of GTP, suggesting that the increased labelling was not due to elevation of the level of beta gamma-subunits or increase in the concentration of GTP in the membranes of noradrenaline-treated cells. Analysis of the PTX substrates on high resolution urea/SDS-polyacrylamide gels revealed that at least two distinct PTX substrates (40 and 41 kDa) were present in rat heart cell membranes. The labelling of both substrates was increased in membranes of desensitized cells. Immunoblotting of heart cell membranes with anti-Gi alpha-antibodies demonstrated a marked increase in the amount of Gi alpha in membranes of noradrenaline-treated cells. In contrast, immunoblotting with anti-beta-antibodies showed that the level of the beta-subunit of G-proteins (36 kDa) was unchanged after noradrenaline exposure. The data indicate that prolonged treatment of rat heart muscle cells with noradrenaline leads to an increase in the level of alpha-subunits of Gi-proteins. This suggests that this increase is responsible for the observed heterologous desensitization of adenylate cyclase stimulation.     

Inhibitory effects of cadmium on in vitro calcification of a clonal osteogenic cell, MC3T3-E1

To examine an inhibitory mechanism of Cd on bone formation, the effects of Cd on calcification were investigated in a culture of a clonal osteogenic cell line, MC3T3-E1. At 3 days after inoculation, Cd was added to the medium containing 7 mM beta-glycerophosphate, and culture was continued for 8 days. Cd at 1.78 microM and above caused a significant decrease in 45Ca accumulation. The decrease in mineralization by Cd was similar to that in collagen content or alkaline phosphatase (ALP) activity. Histologically, the cell density and the mineralization degree were lower than those of the controls. Ultrastructurally, degenerated cells were observed with undifferentiated cells which had fewer rough-surfaced endoplasmic reticulum and many mitochondria. This suggests that Cd may inhibit the differentiation into osteoblasts as well as the cell function. On the other hand, calcification of cells at 8 days after inoculation was inhibited by Cd at 1.78 microM and above. The decrease in collagen content and ALP activity by Cd was much lower than that in calcification. Cd-treated cells were well differentiated into osteoblasts morphologically, but the mineralization degree was lower than that of the controls. Ultrastructurally, cell damage was not recognized so strongly compared with long-term Cd treatment. The mineralization of osteoblasts was also inhibited by Zn levels which left both collagen content and ALP activity unaffected. From these results, it was suggested that the inhibitory effect of Cd on in vitro calcification of MC3T3-E1 cells may be due to both a depression of cell-mediated calcification and a decrease in physiochemical mineral deposition.     

Effect of cadmium and other metal cations on in vitro Leydig cell testosterone production.

In vivo assessment of toxicant action on Leydig cell function is subject to homeostatic mechanisms which make it difficult to determine whether any changes seen in serum testosterone (T) concentration are due to extragonadal endocrine alterations or to a direct effect on the Leydig cell. For example, metal cations administered in vivo have been shown to depress serum T concentration and alter serum concentrations of pituitary hormones in laboratory animals. The studies reported here use a testicular cell culture technique to evaluate Leydig cell testosterone biosynthesis in the presence of several metal cations. To determine the site of toxic action, the Leydig cells were stimulated to produce testosterone by using human chorionic gonadotrophin (hCG), dibutyl cyclic adenosine monophosphate (db-cAMP), or several substrates required for the biosynthesis of testosterone. hCG was chosen because resultant T production requires an intact membrane receptor and db-cAMP was used to test for post LH receptor defects caused by the metals. The other substrates were chosen to isolate the effect of metals on enzymatic pathways. Collagenase dispersed testicular cells (15% Leydig cells) were incubated with metal cations (1 to 5000 microM) for 3 hr in the absence and presence of maximally stimulating concentrations of hCG, db-cAMP, 20 alpha-hydroxycholesterol (HCHOL), or pregnenolone (PREG), and T concentration was determined by radioimmunoassay. In one separate experiment we also tested the effect of the substrates progesterone, 17 alpha-hydroxy-progesterone, and androstenedione on Cd2(+)-treated Leydig cells. The results show no change in Leydig cell viability with any metal cation treatment during the 3-hr incubation. Ca2+, Cr3+, Fe3+, Mg2+, Na+, or Pb2+ had no effect on stimulated testosterone. Dose-response depression in both hCG- and db-cAMP-stimulated T production were seen with Cd2+, Co2+, Cu2+, Hg2+, Ni2+, and Zn2+ treatment. Surprisingly, Cd2+, Co2+, Ni2+, and Zn2+, which caused a depression in hCG- and db-cAMP-stimulated T production, caused significant increases in HCHOL- and PREG-stimulated T production over untreated and similarly stimulated cultures. This indicates that these cations may act at multiple sites within the Leydig cell.     

Mechanism of noradrenaline-induced heterologous desensitization of adenylate cyclase stimulation in rat heart muscle cells: increase in the level of inhibitory G-protein α-subunits

The mechanism of heterologous desensitization of adenylate cyclase stimulation was studied in cultured neonatal rat heart muscle cells. After culturing of the cells for 3 days in the presence of 1 μM noradrenaline there was in addition to a 52% decrease in isoproterenol-stimulated adenylate cyclase activity, a lessening of the stimulation of ß-adrenoceptor-independent adenylate cyclase by guanosine-5′-O-(thiotriphosphate) and forskolin by 24 and 34%, respectively. The decrease in receptor-independent adenylate cyclase stimulation by forskolin, but not the attenuation of isoproterenol-stimulated adenylate cyclase activity, was abolished by pertussis toxin (PTX) pretreatment of the cells. G_i, the inhibitory G-protein of adenylate cyclase was therefore quantitated. Labelling of the M_r ≈ 40 kDa PTX substrates in membranes of noradrenaline-treated cells was increased by 70% as shown by pertussis toxin-catalyzed ADP ribosylation of heart cell membranes. This increase was also seen in the presence of an excess of purified ßγ-subunits of transducin and of GTP, suggesting that the increased labelling was not due to elevation of the level of ßγ-subunits or increase in the concentration of GTP in the membranes of noradrenaline-treated cells. Analysis of the PTX substrates on high resolution urea/SDS-polyacrylamide gels revealed that at least two distinct PTX substrates (40 and 41 kDa) were present in rat heart cell membranes. The labelling of both substrates was increased in membranes of desensitized cells. Immunoblotting of heart cell membranes with anti-G_iα-antibodies demonstrated a marked increase in the amount of G_iα in membranes of noradrenaline-treated cells. In contrast, immunoblotting with anti-ß-antibodies showed that the level of the ß-subunit of G-proteins (36 kDa) was unchanged after noradrenaline exposure. The data indicate that prolonged treatment of rat heart muscle cells with noradrenaline leads to an increase in the level of α-subunits of G_i-proteins. This suggests that this increase is responsible for the observed heterologous desensitization of adenylate cyclase stimulation.     

Copper,but not cadmium,is acutely toxic for trout hepatocytes: short-term effects on energetics and ion homeostasis

The toxic effects of cadmium (Cd) and copper (Cu) on cellular energy metabolism and ion homeostasis were investigated in hepatocytes from the rainbow trout, Oncorhynchus mykiss. The metal content of cells did not increase during incubation with Cu, whereas a dose-dependent increase was seen with Cd. Cell viability was unaffected in the presence of 100 microM Cd and 10 microM Cu but was significantly reduced after 30 min of exposure to 100 microM Cu, both in the presence and absence of extracellular calcium. Oxygen consumption (VO(2)) was not affected by 100 microM Cd or 10 microM Cu, whereas 100 microM Cu caused a significant and calcium-dependent increase of VO(2). Lactate production and basal glucose release were not altered by either of the metals. However, the epinephrine-stimulated rate of glucose release was significantly reduced after 2 h of incubation with 100 microM Cu. Hepatocytes exposed to Cd showed only a marginal increase of intracellular free calcium (Ca(i)(2+)), whereas with Cu a pronounced and dose-dependent increase of Ca(i)(2+) was induced after a delay of 10 to 15 min, the calcium being of extracellular origin. Intracellular pH was not altered by Cd but decreased significantly in the presence of Cu. Overall our data demonstrate that Cu, but not Cd, is acutely toxic for trout hepatocytes. Since Cu does not enter the cells in the short term it appears to exert its acutely toxic effects at the cell membrane. Although Cu toxicity is associated with an uptake of calcium from extracellular space, leading to an elevation of cellular respiration, cytotoxicity does not appear to be dependent on the presence of extracellular calcium.     

Immunomodulation of in vitro antigen presentation by cations

The ability of various cations to alter an antigen-specific (ovalbumin) T cell activation system in vitro has been assessed. This in vitro system includes analysis of antigen capture, processing, and presentation by antigen presenting cells (APC) (splenocytes, B cells or macrophages) in a major histocompatibility complex (MHC) restricted fashion and the evaluation of the activation of a T-cell hybridoma, DO-11.10, which is specific for "processed" ovalbumin in association with I-Ad. Activation is determined by production of IL2 which is quantitated in a bioassay with HT-2 cells. Numerous metals (10 and 100 microM) were screened in the coculture assay (APC and DO-11.10 plus ovalbumin). Metals with inhibitory effects were Cd greater than Cu greater than Pb greater than Zn. Co and Cr had no modulatory effect and Ni had an enhancing effect (increased IL2 production). The effects of the modulatory metals were further assessed for influences on the individual cellular components of this system. Cd was toxic to all cell types whereas Cu was toxic only to irradiated splenocytes and Do-11.10. Pb was the only metal which was not toxic to any cell type but still inhibited antigen presentation. To prevent influences of metals on DO-11.10 or carryover into the bioassay, APC were preincubated with metals and then assessed for presentation capability. After preincubation, only Cd, Pb, and Cu were inhibitory. The inhibition by Cd was due to toxicity. Cu inhibited only irradiated splenocyte presentation and this effect was due to toxicity. Pb inhibition was due to a mechanism other than toxicity and its biochemical influences on APC are discussed.     

Histological and clinical studies on the effects of low to medium level infrared light therapy on human and mouse skin

BACKGROUND AND OBJECTIVE: Deep heating or denaturation of collagen has been reported to be necessary for nonablative skin rejuvenation. The purpose of this study was to examine whether thermally damaged collagen is an indispensable factor to increase the amount of collagen in vivo. Epidermal and dermal responses to infrared light therapy using a Titan source were examined with the aim of correlating histological and clinical responses in human and amelanotic mouse skin. STUDY DESIGN/MATERIALS AND METHODS: Ten, 20, or 30 J/cm2 infrared light were irradiated on the human subject's skin (thigh), while 5, 10, 20, or 30 J/cm2 were used on amelanotic mouse skin. Biopsies were taken and analyzed using hematoxylin and eosin (H&E) and Elastica von Gieson stain. RESULTS: Ten or 20 J/cm2 infrared light increased the amount of both collagen and elastin in all layers of the dermis without denaturing the collagen in human skin. A higher dose of 30 J/cm2 also increased the amount of collagen and elastin, but denatured the collagen in human skin. (In addition to the thigh, 2 treatments of 10 J/cm2 infrared light improved skin toning and texture on the subject's face). In mouse skin, 5 or 10 J/cm2 remarkably increased the amount of both collagen and elastin, and of epidermal cells. Twenty or 30 J/cm increased the amount of collagen and elastin and the number of keratinocytes, but caused some vacuolated degeneration of keratinocytes. The presence of denatured collagen was not evident due to the high density of collagen. CONCLUSIONS: This study shows that the denaturation of collagen is not required to increase the amounts of collagen or elastin in vivo in human skin. The activation of the mitochondria as well as the denaturation of collagen may play important roles in infrared phototherapy.     

Comparison of the effects of semicarbazide and β-aminopropionitrile on the arterial extracellular matrix in the Brown Norway rat

To investigate a putative role for semicarbazide-sensitive amine oxidase (SSAO) in arterial extracellular matrix (ECM) organization, we compared arteries of growing Brown Norway (BN) rats after chronic administration of semicarbazide (SCZ) and β-aminopropionitrile (BAPN), two inhibitors with different properties and relative specificities for SSAO and lysyl oxidase (LOX). The BN model is particularly well adapted to evaluating effects of toxic compounds on the arterial elastic network. We measured aortic LOX and SSAO activities and quantified several ECM parameters. After a pilot study comparing doses previously studied and testing for additivity, we studied low and high equimolar doses of SCZ and BAPN. Both compounds similarly inhibited LOX, whereas SCZ inhibited SSAO far more effectively than BAPN. Both decreased carotid wall rupture pressure, increased tail tendon collagen solubility, decreased aortic insoluble elastin (% dry weight) and dose-dependently increased defects in the internal elastic lamina of abdominal aorta, iliac and renal arteries. Our results suggest that either these effects are mediated by LOX inhibition, SCZ being slightly more effective than BAPN in our conditions, or SSAO acts similarly to and in synergy with LOX on ECM, the greater SCZ effect reflecting the simultaneous inhibition of both enzymes. However, the high SCZ dose increased aortic collagen and ECM proteins other than insoluble elastin markedly more than did equimolar BAPN, possibly revealing a specific effect of SSAO inhibition. To discriminate between the two above possibilities, and to demonstrate unequivocally a specific effect of SSAO inhibition on ECM formation or organization, we must await availability of more specific inhibitors.