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Abstract
The effect of a range of concentrations of ethanol on collagen biosynthesis in vitro was studied by measuring 14C-proline uptake and 14C-hydroxyproline production in embryonic chick tibia preparations. With increasing ethanol concentration there was decreasing proline incorporation. Hydroxyproline synthesis was relatively stimulated by low, but was progressively inhibited by higher, concentrations of ethanol. The effect of certain proprietary alcohols in this simple experimental preparation did not correlate with their ethanol content. The evidence indicates that certain of the congeners in alcoholic beverages are potentially cirrhogenic.
... A dense, collagen-rich mass co-localizes with the proliferation of tumor cells (Kao et al., 1986; Alowami et al., 2003; Li et al., 2005; Schedin and Keely, 2011), even if the actual production of collagen takes place in stromal cells rather than in tumor cells. Cross-regulation between collagen synthesis and tumor cell proliferation may be mediated by mechanoregulatory (Provenzano et al., 2008 ) or metabolic reprogramming mechanisms (Walker and Shand, 1972; Savolainen et al., 1984; Anthony et al., 2010). The latter suggestion is plausible because metabolic abnormalities concomitant to inflammatory proliferation is frequently associated with collagen formation, i.e., fibrosis (Davis and Cowie, 1990; Eddy and Giachelli, 1995). ...
Studies in metabolism and cancer have characterized changes in core pathways involving glucose and glutamine, emphasizing the provision of substrates for building cell mass. But recent findings suggest that pathways previously considered peripheral may play a critical role providing mechanisms for cell regulation. Several of these mechanisms involve the metabolism of non-essential amino acids, for example, the channeling of glycolytic intermediates into the serine pathway for one-carbon transfers. Historically, we proposed that the proline biosynthetic pathway participated in a metabolic interlock with glucose metabolism. The discovery that proline degradation is activated by p53 directed our attention to the initiation of apoptosis by proline oxidase/dehydrogenase. Now, however, we find that the biosynthetic mechanisms and the metabolic interlock may depend on the pathway from glutamine to proline, and it is markedly activated by the oncogene MYC. These findings add a new dimension to the proline regulatory axis in cancer and present attractive potential targets for cancer treatment.
... A dense, collagen-rich mass co-localizes with the proliferation of tumor cells (Kao et al., 1986;Alowami et al., 2003;Li et al., 2005;Schedin and Keely, 2011), even if the actual production of collagen takes place in stromal cells rather than in tumor cells. Cross-regulation between collagen synthesis and tumor cell proliferation may be mediated by mechanoregulatory (Provenzano et al., 2008) or metabolic reprogramming mechanisms (Walker and Shand, 1972;Savolainen et al., 1984;Anthony et al., 2010). The latter suggestion is plausible because metabolic abnormalities concomitant to inflammatory proliferation is frequently associated with collagen formation, i.e., fibrosis (Davis and Cowie, 1990;Eddy and Giachelli, 1995). ...
Proline plays a special role in cancer metabolism. Proline oxidase (POX), a.k.a. proline dehydrogenase (PRODH), is among a few genes induced rapidly and robustly by P53, the tumor suppressor. Ectopic expression of POX under control of tet-off promoter initiated mitochondrial apoptosis. The mechanism activated by POX is mediated by its production of ROS. In immunodeficient mice, POX overexpression markedly retarded growth of xenograft tumors. In human tumors of the digestive tract and kidney, POX was markedly decreased, suggesting that the suppressive effect of POX was downregulated. This was not due to POX gene mutations or hypermethylation. Instead, a microRNA, miR-23b*, expressed at high levels in tumors, was a potent inhibitor of POX expression. Furthermore, antagomirs of miR-23b* reversed the downregulated expression of POX and its tumor-suppressive effect, thereby providing a therapeutic strategy. POX not only responds to genotoxic stress, but also to inflammatory and metabolic stress. Depending on microenvironmental and temporal factors, POX can mediate oppositely-directed responses-programmed cell death, on the one hand, and survival, on the other.
The mortality rates for cirrhosis vary greatly from country to country. For example, in 1972 the World Health Organization reported mortality rates of 7.5/100,000 in Finland and 57.2/100,000 in France. The corresponding per-capita alcohol consumptions were 5.1 and 16.8 liters of absolute alcohol (International Statistics on Alcoholic Beverages: Production Trade and Consumption 1950–1972, 1977). Whereas deaths from other causes such as cardiovascular diseases decreased in the period from 1950 to 1974 by 2% in the United States, deaths from cirrhosis climbed 71.7%. Since then, mortality from cirrhosis has leveled off or even decreased in the United States. The reduction, however, pertains primarily to nonalcoholic cirrhosis (Liver Cirrhosis Mortality in the United States, 1988). In Canada, cirrhosis has been the most rapidly increasing cause of death in the population over 25 years of age, followed by lung and bronchial cancer and suicide (Schmidt, 1977), and it is now the fifth major cause of death for men in the productive years from 25 to 64 (Rankin, 1977). The concentration of mortality of alcoholic cirrhosis to a rather young population group also occurs in the United States. In Baltimore, it was the fifth leading cause of death for persons 25 to 44 years of age (Kuller et al., 1969) and the fourth leading cause of death for those 25 to 64 years of age in New York City (New York City; Summary of Vital Statistics, 1984).
Alcohol problems are common in nonalcoholic young people, and alcoholism is one of the most frequently encountered diseases in clinical practice. Specific dental conditions and pathoses associated with alcohol consumption are reviewed. Approximately a third of dental patients can be expected to show minor alcohol-related problems, and 10% to 25% of the patients might have such severe alcohol problems that they could be labeled as alcoholics. This paper reviews alcoholism and the way in which alcohol-related conditions can affect the clinical situation.
To assess the value of type III procollagen peptide (sPIIIP) as a marker of hepatic fibrosis, sera from 73 patients with alcohol-related liver disease and 30 patients with idiopathic hemochromatosis (IHC) were studied by a specific radioimmunoassay. sPIIIP was increased in 87% of 30 patients with cirrhosis, in 16% of 32 with steatofibrosis but in none of 11 with steatosis.
There was a significant correlation with histologic hepatocellular necroinflammation (r = 0.42, p < 0.01), but not with hepatic fibrosis.
sPIIP was increased in 33% of 30 patients with IHC, whether or not they had cirrhosis or fibrosis, and whatever the level of iron overload or the extent of the hepatic deterioration. IHC patients with increased levels of sPIIIP had a higher prevalence of superimposed hepatic damage than did those with normal procollagen levels (p < 0.05).
Our findings, therefore, weaken the diagnostic value of sPIIIP as an index of connective tissue deposition in the liver, and suggest that, at least in alcohol-related liver disease and IHC, hepatocellular necroinflammation influences the results.
Serum free proline and free hydroxyproline were determined in 71 patients with liver disease and in 62 control subjects. The group with liver disease included 60 patients with liver cirrhosis and 11 with chronic active liver disease. Forty-five of the cirrhotic patients were alcoholics, 9 of which were studied during an episode of alcoholic hepatitis. The control group consisted of 24 healthy volunteers, 19 primary malnourished patients, and 19 severely ill patients without liver disease. The values obtained in normal subjects were quite constant; no differences related to sex or age were detected. In patients with malnutrition, and especially in severely ill patients, the proline values were always below the normal limit. Patients with nonalcoholic cirrhosis or chronic active liver disease had serum proline and hydroxyproline values similar to those of normal subjects. However, the patients with alcoholic liver cirrhosis had proline and hydroxyproline values significantly higher than the normal group. Furthermore, in patients with alcoholic hepatitis the serum free proline values were significantly higher than in the other groups. The results suggest that alcohol might have a direct effect on proline metabolism or facilitate its release from the liver cell.
Percutaneous liver biopsies obtained from patients with a history of chronic alcoholism and normal liver, fatty liver, alcoholic hepatitis, or active cirrhosis were incubated with tritiated proline to determine the pattern of collagen biosynthesis in these conditions. Incorporation of labeled proline and hydroxyproline into salt-soluble and insoluble fractions of collagen was evaluated by radiochemical analysis and tissue localization documented by autoradiography. Biopsy specimens of alcoholic hepatitis and cirrhosis exhibit a significant increase in the amount of radioactive proline and hydroxyproline in salt-soluble and insoluble collagen. Marked accumulation of radioactivity occurred over bile ducts, fibroblasts, and collagen fibers in the portal area and over hepatocytes, fibroblasts, and collagen fibers in the centrilobular area. Fatty liver is associated with an increase in uptake of proline and hydroxyproline in the salt-soluble fraction of collagem; silver grains appear in the periphery of fat-laden cells and in areas of focal inflammation. Digestion by collagenase indicates that labeling over fibroblasts and collagen reflects active synthesis, whereas, entry of proline into the cell protein pool is responsible for accumulation of radioactivity in other sites. In vitro ethanol causes a significant increase in the incorporation of proline and hydroxyproline into collagen in biopsy specimens of alcoholic hepatitis or active cirrhosis, but has no effect on collagen synthesis by normal or fatty liver.
Ethanol is hepatotoxic through redox changes produced by the NADH generated in its oxidation via the alcohol dehydrogenase pathway, which in turn affects the metabolism of lipids, carbohydrates, proteins and purines. Ethanol is also oxidized in liver microsomes by an ethanol-inducible cytochrome P-450 (P-450IIE1) which contributes to ethanol metabolism and tolerance, and activates xenobiotics to toxic radicals thereby explaining increased vulnerability of the heavy drinker to industrial solvents, anesthetic agents, commonly prescribed drugs, over-the-counter analgesics, chemical carcinogens and even nutritional factors such as vitamin A. Induction also results in energy wastage and increased production of acetaldehyde. Acetaldehyde, in turn, causes injury through the formation of protein adducts, resulting in antibody production, enzyme inactivation, decreased DNA repair, and alterations in microtubules, plasma membranes and mitochondria with a striking impairment of oxygen utilization. Acetaldehyde also causes glutathione depletion and lipid peroxidation, and stimulates hepatic collagen synthesis, thereby promoting fibrosis.
Sake, a rice wine, induced hepatic collagen accumulation in rats. This was noted after 16 weeks of a liquid diet containing 35% ethanol by caloric content. However, under similar experimental conditions, whiskey and ethanol did not produce any changes. Hepatic collagenase activities in sake-fed rats were slightly, but significantly, higher than in the whiskey group. The central and pericellular liver fibrosis in sake-fed rats was caused possibly by either accelerated collagen synthesis or maturation, exceeding the increased collagen degradation. Mechanisms of enhanced fibrogenesis in sake-fed rats were discussed.
From in-vitro lymphocyte transformation in response to stimulation by Australia antigen, evidence of cell-mediated immunity to hepatitis virus B has been found in all of eleven cases of chronic alcoholic liver disease. It seems likely that infection by this virus is important in the aetiology of this disease.
Cultures of liver cells (Chang) and of fibroblasts (3T6) were exposed to media containing a range of concentrations of ethanol. At the end of a standard period of time the number of viable cells was determined. As the concentration of ethanol increased over 0.25 per cent v/v liver cell survival decreased progressively. Fibroblast survival decreased progressively at ethanol concentrations over 0.50 per cent v/v. This indicates that ethanol is more toxic to liver cells than to fibroblasts. Certain alcoholic beverages, particularly brandy, were found to be more toxic than pure ethanol.
Addition of ethanol to cultured fetal lung fibroblasts resulted in decreases of both collagen and noncollagen protein syntheses. The inhibitory effect of ethanol on protein synthesis was dependent on the concentration of ethanol and the number of treatments with ethanol. Significant inhibition of collagen and noncollagen protein synthesis was observed 3 hr after a single treatment in 0.25% (v/v) ethanol. The maximum inhibitory effect of ethanol on protein synthesis was observed at 6 hr after drug addition. Inhibition of protein synthesis was observed when either proline or glycine was used as the precursor amino acid. An inhibition of alcohol dehydrogenase did not block the ethanol-mediated inhibition of protein synthesis. Ethanol, added to cell cultures throughout the log phase, inhibited cell growth during the late log and stationary phases. Ethanol inhibition of collagen and noncollagen protein synthesis was reversed when the cell cultures were washed and suspended in fresh media for 24 hr. These inhibitory effects of ethanol on macromolecular syntheses were not engendered by killing of cells. The viability of the cells, as indicated by trypan blue exclusion, was not affected significantly at the concentrations of ethanol used. The inhibitory effect of ethanol on protein synthesis also did not originate from drug-mediated inhibition of precursor amino acid uptake. Polysomes isolated from ethanol-treated fibroblasts incorporated proline into protein at a rate which was reduced commensurate with cellular protein synthesis. The resultant inhibition by ethanol of protein synthesis was not attributable to a direct effect of drug on polysomes. Treatment of fetal lung fibroblasts with ethanol also caused a marked inhibition of radioactive thymidine and uridine incorporation, indicating a reduction of both total cellular DNA and RNA synthesis. Accordingly, the decrease of protein synthesis resulted from inhibition of RNA synthesis. Furthermore, messenger RNA synthesis may have decreased since polysomes isolated from ethanol-treated fibroblasts synthesized less protein in the wheat germ cell-free system. Unlike other biochemical variables that were inhibited by ethanol treatment, the level of prolyl hydroxylase activity was elevated significantly. The elevated level of prolyl hydroxylase activity, however, was related neither to the rate of collagen polypeptide synthesis nor to the degree of proline hydroxylation of cellular collagen. The data suggest that the growth-retarding effects of nonlethal doses of ethanol on fetal development may result from inhibition of macromolecular synthesis in fetal fibroblasts.
Continuing hepatic injury despite cessation of ethanol intake has suggested a potential role for altered humoral and cellular immunity in the pathogenesis of alcoholic liver disease. Variation of cell-mediated response has been observed most commonly in patients with alcoholic hepatitis. These alterations include decreased mitogen response in vitro, reduction of circulating T-cell numbers with a corresponding increase of intrahepatic T-cells, lymphokine production in vitro in response to isolated alcoholic hyalin (Mallory bodies), and in vitro evidence of increased spontaneous cell-mediated cytotoxicity. Fewer immunologic alterations are detected in patients with alcoholic fatty liver or cirrhosis. At present, these observations have not established a definite causal relationship between ethanol-induced liver injury and immunologic mechanisms. Studies utilizing in vitro correlates of cellular immunity in alcoholics are carried out after development of hepatic injury. To establish the role of humoral and cellular immune events in the pathogenesis of alcoholic liver injury will require development of a suitable animal model.
Cells with electron-microscopic characteristics of myofibroblasts were isolated from baboon liver biopsy specimens by collagenase digestion and Percoll density gradient centrifugation and then cultured. The cultures consisted of only one cell type. By immunofluorescence, these cells synthesized collagen types I, III, and IV and laminin. Typical features of myofibroblasts were maintained throughout many passages in the culture. To study the effects of ethanol (and its oxidation product acetaldehyde and associated metabolite lactate) on myofibroblast collagen synthesis, the cell cultures were incubated for 24 h in a medium containing either 50 mM ethanol, 200 microM acetaldehyde, or 5 mM lactate. The cells did not contain significant alcohol dehydrogenase activity. Acetaldehyde stimulated significantly (p less than 0.05) myofibroblast collagen synthesis without changing noncollagen protein synthesis or proline pools. Lactate caused a significant (p less than 0.02) increase in intracellular proline pool and collagen synthesis. Ethanol itself did not have any effect on collagen synthesis of myofibroblasts. The stimulation of collagen synthesis of hepatic myofibroblasts by acetaldehyde and lactate may contribute to the development of alcoholic liver fibrosis, as alcohol intake is known to elevate acetaldehyde and lactate in tissues and blood.
In this study bone morphometric findings in 19 heavy or moderate drinkers are compared with those in 43 non-drinkers (22 non-osteoporotic patients with osteo-arthritis (OA) and 21 osteoporotic patients with femoral neck fractures (FNFs). Transiliac bone biopsy specimens were examined for assessment of trabecular bone volume and thickness, bone resorption and formation, osteoid seam length and width and the mineralization front by quantitative histomorphometry and microradiography. Trabecular bone volume and thickness were significantly diminished in the drinking group when compared with those in the 22 non-drinking patients with OA (P less than 0,005 and P less than 0,0001 respectively). Bone formation was significantly lower in the drinkers than in the OA group (P less than 0,0005), and bone resorption was significantly greater in the drinkers than in the OA (P less than 0,005) and FNF (P less than 0,01) groups. In heavy drinkers bone formation was significantly lower (P less than 0,05) than in moderate drinkers. The extent of the mineralization front was significantly lower in the drinking group than in the OA (P less than 0,005) and FNF (P less than 0,0005) groups. Osteoid seam length and width did not differ in the three groups. The presence of abnormal liver function test results did not significantly affect any of the parameters. It is concluded that alcohol abuse is associated with: (i) uncoupling of bone resorption and bone formation; (ii) reduced trabecular bone volume and thickness; and (iii) a mild mineralization defect.
A toxic effect of alcohol is the principal cause of the development of liver disease in alcoholism. Fatty infiltration of the liver is a consequence of ethanol metabolism due mainly to an increased synthesis and decreased degradation of fatty acids. Mechanisms that have been suggested for ethanol-induced hepatocellular necrosis include centrolobular hypoxia due to an increased oxygen requirement and intracellular accumulation of protein, fat, and water which results in increased cell size. Hepatocellular necrosis, however, may not be a necessary stage in the development of cirrhosis. Chronic ethanol administration increases hepatic collagen deposition, and acute and chronic ethanol administration inhibit liver cell regeneration. Increased humoral and cellular immunological activity to liver tissue and its components may contribute to the persistence of liver disease in the alcoholic. However, only a small proportion of alcoholics and baboons fed alcohol develop cirrhosis, suggesting that other factors, either genetic, environmental, or nutritional, play a role. Malnutrition is common in alcoholics. Liver disease is more common in some malnourished populations, and has been produced by nutrient deficiencies. Decreased dietary intake, as well as malabsorption and alterations in the metabolism of nutrients, are causes of nutrient deficiencies in alcoholism. Some of the effects of alcohol on the liver may be mediated by its actions on nutrient absorption and metabolism.
In a postmortem exploratory study, we examined whether specific amino acid abnormalities associated with liver diseases in vivo may also be detected in human brain samples obtained at clinical autopsies. The branched-chain amino acids (BCAA: valine, leucine, isoleucine) were decreased in the group of patients with liver diseases compared with the control group, whereas the aromatic amino acids (AAA: phenylalanine, tyrosine) were increased. However, the ranges overlapped significantly and were not statistically different. The molar ratio BCAA/AAA was determined to be 1.92 in the collection of patients with liver diseases compared with 2.27 in the control group. In patients with liver disease, ornithine concentrations in the brain appeared significantly decreased whereas glutamine was significantly increased. No significant difference was found in the brain concentrations of proline. Amino acid analysis may contribute to the understanding of pathophysiological mechanisms of liver disease, which are discussed, and may supplement the postmortem diagnosis.
A prospective clinical study was conducted to explore the possible link between peri-implant bone loss and the widespread habits of tobacco smoking and alcohol consumption. One hundred and eighty-five patients who received 514 implants were followed up for 3 years. Peri-implant marginal bone loss was evaluated by digital panoramic radiography and image analysis techniques. Multivariate analysis showed that peri-implant marginal bone loss was significantly related to a daily consumption of >10 g of alcohol, tobacco use and increased plaque levels and gingival inflammation. The present results indicate that daily alcohol consumption and tobacco use may have a negative influence on predictable long-term implant treatment outcomes, producing peri-implant bone loss and compromising restorative treatment with implant-supported prostheses.
Ultrastructural and biochemical observations in hepatic fibrosis have been reviewed. Recent advances in our knowledge of collage metabolism have helped in understanding the kinetics of fibrosis in the hepatic parenchyma. Instead of fiber accumulation from collapse previously emphasized, new formation of fibers is now considered of major importance. Three processes have to be distinguished, namely, stimulation of fibroblasts, maturation and deposition of fibers, and catabolism of fibers. These considerations may eventually become useful in development of a rational therapy of fibrosis and cirrhosis, particularly since some agents like corticosteroids may have antagonistic effects on the three stages. Hepatic fibrosis probably stimulated by hepatocellular injury may, in turn, damage the hepatic parenchyma, and this may be more important in the self-perpetuation of liver injury than the immunologic components of hepatic inflammation. This damage is produced by local interference with hepatocellular nutrition and by some of the hemodynamic alterations characteristic of cirrhosis which are exerted by the formation of septums. The observations presented may also be useful in developing clinical parameters for determining the extent of active hepatic fibrogenesis.
The optimal conditions for hydrolysis of rat skin for maximal yield of hydroxyproline were determined:4 N HCl at a temperature of 110° C for 22 hours in Teflon-lined screw-capped tube.
The spectrophotometry method of Bergman and Loxley for the determination of hydroxyproline based on oxidation by Chloramine T was modified to give reproducible results with considerable savings of time.
Evaporation of the acid hydrolysate was not attempted, because even neutralization of the hydrolysate was proved unnecessary in the present scheme. The final dilution step using isopropanol before color measurement was omitted.
However, the “salt effect” of NaCl on the absorbance of treated hydroxyproline solution was studied; no effect was detected until NaCl reached 0.6 M. This information is important for investigators employing hypertonic salt solution for the extraction of neutral soluble collagen from biological tissues.
Glycine and proline, which are the other two major constituents of collagens were found not to interfere with the hydroxyproline analysis. Hydroxyproline by itself and hydroxyproline added to tissue were recovered quantitatively in recovery studies, indicating no destruction of this imino acid under the hydrolyzing conditions. The purity of spectra of the developed color for unknown solutions was demonstrated.