Onni Niemelä

Distribution of ethanol-induced protein adducts in vivo: relationship to tissue injury.

Generation of oxygen free radicals and reactive aldehydes as a result of excessive ethanol consumption has been well established. Recent studies in human alcoholics and in experimental animal models have indicated that acetaldehyde, the first metabolite of ethanol, and the aldehydic products of lipid peroxidation can bind to proteins in tissues forming stable adducts. The demonstration of such adducts in zone 3 hepatocytes in alcoholics with an early phase of histological liver damage indicates that adduct formation may have an important role in the sequence of events leading to alcoholic liver disease. There may be interference with cellular functions, stimulation of fibrogenesis, and immunological responses. Autoantibodies towards distinct types of adducts have been shown to be associated with the severity of liver disease in alcoholic patients. High fat diet and/or iron supplementation combined with ethanol may increase the amount of aldehyde-derived epitopes and promote fibrogenesis in the liver. Recently, ethanol-derived protein modifications have also been found from other tissues exposed to ethanol and acetaldehyde, including rat brain after lifelong ethanol administration, pancreas, and rat muscle. Elevated adduct levels also occur in erythrocytes of alcoholics, which may be related to ethanol-induced morphological aberrations in hematopoiesis.

Folate deficiency disturbs hepatic methionine metabolism and promotes liver injury in the ethanol-fed micropig

Alcoholic liver disease is associated with abnormal hepatic methionine metabolism and folate deficiency. Because folate is integral to the methionine cycle, its deficiency could promote alcoholic liver disease by enhancing ethanol-induced perturbations of hepatic methionine metabolism and DNA damage. We grouped 24 juvenile micropigs to receive folate-sufficient (FS) or folate-depleted (FD) diets or the same diets containing 40% of energy as ethanol (FSE and FDE) for 14 wk, and the significance of differences among the groups was determined by ANOVA. Plasma homocysteine levels were increased in all experimental groups from 6 wk onward and were greatest in FDE. Ethanol feeding reduced liver methionine synthase activity, S-adenosylmethionine (SAM), and glutathione, and elevated plasma malondialdehyde (MDA) and alanine transaminase. Folate deficiency decreased liver folate levels and increased global DNA hypomethylation. Ethanol feeding and folate deficiency acted together to decrease the liver SAM/S-adenosylhomocysteine (SAH) ratio and to increase liver SAH, DNA strand breaks, urinary 8-oxo-2′-deoxyguanosine [oxo(8)dG]/mg of creatinine, plasma homocysteine, and aspartate transaminase by more than 8-fold. Liver SAM correlated positively with glutathione, which correlated negatively with plasma MDA and urinary oxo(8)dG. Liver SAM/SAH correlated negatively with DNA strand breaks, which correlated with urinary oxo(8)dG. Livers from ethanol-fed animals showed increased centrilobular CYP2E1 and protein adducts with acetaldehyde and MDA. Steatohepatitis occurred in five of six pigs in FDE but not in the other groups. In summary, folate deficiency enhances perturbations in hepatic methionine metabolism and DNA damage while promoting alcoholic liver injury.

Cytochromes P450 2A6, 2E1, and 3A and production of protein-aldehyde adducts in the liver of patients with alcoholic and non-alcoholic liver diseases

BACKGROUND/AIMS Interaction between CYP2E1, ethanol metabolites, and enhanced lipid peroxidation is linked to the pathogenesis of alcoholic liver disease. This study was conducted to compare the expression of various cytochrome enzymes and the appearance of aldehyde adducts in humans. METHODS Acetaldehyde- and lipid peroxidation-derived protein adducts and CYP2A6, 2E1, and 3A4/5 were examined immunohistochemically from liver specimens of 12 alcohol abusers with either mild (n=7) or severe (n=5) liver disease, and from nine non-drinking patients with non-alcoholic steatosis (n=4), or hepatitis (n=5). RESULTS Ethanol-inducible CYP2E1 was present in all alcoholic livers. While CYP2A6 in zone 3 hepatocytes was also abundant in the alcoholic patients with various degrees of liver disease, CYP3A415 was most prominent in alcoholic cirrhosis. The sites of CYP2E1 and CYP2A6 immunoreactivity co-localized with fatty deposits, and with the sites of acetaldehyde and lipid peroxidation-derived protein adducts. The CYP enzymes were also abundant in the centrilobular hepatocytes of patients with fatty liver due to obesity or diabetes. CONCLUSIONS Alcohol-induced liver damage is associated with a generalized induction of CYP2A6, CYP2E1 and CYP3A4 and generation of acetaldehyde and lipid peroxidation-derived protein-aldehyde adducts. However, CYP induction also occurred in patients with non-alcoholic steatosis.

Roles of oxidative stress in activation of Kupffer and Ito cells in liver fibrogenesis

An increasing body of experimental evidence is emerging to incriminate oxidative stress as a pivotal signal for liver fibrogenesis. This paper reviews the results from our studies testing this hypothesis. In the rat model of alcoholic liver disease, the importance of oxidative stress was supported by marked accentuation of liver fibrosis by dietary supplementation of iron, a pro‐oxidant, and the significant correlation of the liver malondialdehyde (MDA) and 4‐hydroxynonenal (4HNE) levels with the hepatic collagen accumulation. Both MDA and 4HNE adduct epitopes were detected intensely and diffusely in close association with collagen deposition. The direct cause and effect relationship between MDA/4HNE and Ito cell stimulation was indicated by the demonstration of Ito cell collagen gene induction by these aldehydes in culture. In primary cultures of rat Kupffer cells (KC), addition of antioxidants such as α‐tocopherol acetate and succinate suppressed mRNA expression and the release of interleukin (IL)‐6 and tumour necrosis factor alpha (TNFα). In rats with biliary fibrosis, an increase in the liver MDA level was accompanied by enhanced mRNA expression of procollagen α 1 (I) and transforming growth factor β 1 in Ito cells; and that of TNFα and IL‐6 in KC. Furthermore, the gel shift assay of KC nuclear extracts showed enhanced NF‐kB DNA binding activity. These results support the proposal that enhanced oxidative stress constitutes an important signal for activation of Kupffer and Ito cells in experimental liver fibrogenesis.

Type IV collagen and laminin‐related antigens in human serum in alcoholic liver disease

Abstract. The two major constituents of basement membranes are type IV collagen and laminin. Specific radioimmunoassays are described here for two structural domains of these proteins (7‐S collagen and the fragment PI, respectively) that allow the related antigens to be quantified in human serum. The serum 7‐S collagen antigen was uniform in size, whereas the laminin PI antigenicity was heterogeneous. These proteins were measured in sera from sixty‐three alcoholics, divided on the basis of liver histology into four groups: normal light microscopy, fatty liver, alcoholic cirrhosis with hepatitids and inactive cirrhosis. The group with cirrhosis and hepatitis had clearly elevated values in both assays, differing significantly from the others. A few pathological results were also seen in the other groups. The increases noted in 7‐S collagen concentration were larger than those in laminin PI. During follow‐up of a patient with cirrhosis and hepatitis the 7‐S collagen level in particular seemed to reflect the course of the disease. The elevated basement membrane protein concentrations in serum may be associated with the formation of real basement membranes in the perisinusoidal space, a process known as capillarization of the sinusoids which is found during the development of liver cirrhosis.

Markers of Fibrogenesis and Basement Membrane Formation in Alcoholic Liver Disease Relation to Severity, Presence of Hepatitis, and Alcohol Intake

This study investigated the relationships of the serum markers of fibrogenesis and basement membrane formation to the clinical and morphological severity of alcoholic liver disease and to the degree of alcohol abuse. The concentrations of the aminoterminal propeptide of type III collagen, type IV collagen, and laminin were measured from 87 samples representing a wide range of clinical and histological severities of the disease, which were assessed with indices that have been shown to correlate well with the risk of dying within 1 yr. Significant correlations (p less than 0.00001) were found between the markers of connective tissue metabolism and the Combined Clinical and Laboratory Index: (aminoterminal propeptide of type III collagen, rs = 0.82; type IV collagen, rs = 0.82; laminin, rs = 0.81), as well as between these markers and the Combined Morphological Index: (aminoterminal propeptide of type III collagen, rs = 0.70; type IV collagen, rs = 0.68; laminin, rs = 0.64). Whereas the patients with less than 8 mM of alcohol in their morning urine (mild or moderate drinkers) showed a significant (p less than 0.00001) decrease in these markers in a period of 27 +/- 1 wk, the patients with more than 8 mM of urinary alcohol (heavy drinkers) had no improvement. It is proposed that both fibrogenesis and basement membrane formation are associated with disease severity, degree of alcoholic hepatitis, and alcohol intake, which are important determinants of prognosis in alcoholic liver disease.

Timing of infant feeding in relation to childhood asthma and allergic diseases

BACKGROUND Emerging evidence questions current recommendations on the timing of infant feeding for the prevention of childhood allergies. The evidence for asthma is inconclusive. OBJECTIVE We sought to investigate the associations between the duration of breast-feeding and timing of introduction of complementary foods and the development of asthma and allergies by the age of 5 years. METHODS Data were analyzed for 3781 consecutively born children. The dietary exposures were categorized into thirds and analyzed as time-dependent variables. Asthma, allergic rhinitis, and atopic eczema end points were assessed by using the International Study of Asthma and Allergies in Childhood questionnaire, whereas IgE antibodies were analyzed from serum samples at the age of 5 years. Cox proportional hazard and logistic regressions were used for the analyses. RESULTS The median duration of exclusive and total breast-feeding was 1.4 months (interquartile range, 0.2-3.5 months) and 7.0 months (interquartile range, 4.0-11.0 months), respectively. Total breast-feeding of 9.5 months or less was associated with an increased risk of nonatopic asthma. Introduction of wheat, rye, oats, or barley at 5 to 5.5 months was inversely associated with asthma and allergic rhinitis, whereas introduction of other cereals at less than 4.5 months increased the risk of atopic eczema. Introduction of egg at 11 months or less was inversely associated with asthma, allergic rhinitis, and atopic sensitization, whereas introduction of fish at 9 months or less was inversely associated with allergic rhinitis and atopic sensitization. CONCLUSION Early introduction of wheat, rye, oats, and barley cereals; fish; and egg (respective to the timing of introduction of each food) seems to decrease the risk of asthma, allergic rhinitis, and atopic sensitization in childhood. Longer duration of total breast-feeding, rather than its exclusivity, was protective against the development of nonatopic but not atopic asthma, suggesting a potential differing effect of breast-feeding on different asthma phenotypes.

Green areas around homes reduce atopic sensitization in children

Western lifestyle is associated with high prevalence of allergy, asthma and other chronic inflammatory disorders. To explain this association, we tested the ‘biodiversity hypothesis’, which posits that reduced contact of children with environmental biodiversity, including environmental microbiota in natural habitats, has adverse consequences on the assembly of human commensal microbiota and its contribution to immune tolerance.

Immune Responses to Ethanol Metabolites and Cytokine Profiles Differentiate Alcoholics with or without Liver Disease

OBJECTIVES:Excessive alcohol consumption is associated with the generation of antibodies against neoantigens induced by ethanol metabolism. However, the associations between such immune responses, ethanol consumption, and liver injury remain unclear.METHODS:Eight-six male alcoholics with (n = 54) or without (n = 32) liver disease, and 20 male volunteers (6 abstainers, 14 moderate drinkers) underwent clinical, morphological, and biochemical assessments of liver status and ethanol consumption.RESULTS:Antiacetaldehyde adduct IgAs in both groups of alcoholics were significantly higher than those in the controls. Elevated IgGs occurred in patients with liver disease, whereas IgMs were high in the heavy drinkers without apparent liver disease. Liver disease patients had high levels of both proinflammatory (IL-2, IL-6, IL-8, TNF-α) and antiinflammatory (IL-10) cytokines, whereas those without liver disease showed elevated IL-6, IL-8, and IL-10 only. Ethanol consumption correlated significantly with antiadduct IgA and IL-6 levels, which also showed parallel changes upon abstinence.CONCLUSIONS:Alcoholic liver disease is associated with the generation of IgAs and IgGs against acetaldehyde-derived antigens and enhanced levels of both pro- and antiinflammatory cytokines, whereas elevated IgA, IL-6, and IL-10 characterize alcoholics without liver disease. These data suggest that immunological mechanisms may play a role in the sequence of events leading to liver disease in some patients with excessive drinking.

Heterogeneity of the antigens related to the aminoterminal propeptide of type III procollagen in human serum

Inhibition curves that are considerably less steep than the reference peptide curve are a constant finding when human serum samples are studied with the radioimmunoassay for the aminoterminal propeptide Col 1-3 of type III procollagen. This is due to the presence in the serum of three main peptide forms which differ in their antigenic properties and can be separated by gel filtration. Their molecular sizes are, respectively, larger than, equal to and smaller than the peptide Col 1-3. The proportions of these forms were different in a number of serum samples tested. An elevated value in the Col 1-3 radioimmunoassay need not reflect increased deposition of type III collagen in the liver, but could also be due to increased degradation of a newly-synthesized type III procollagen or degradation of a tissue form still containing the aminoterminal propeptide. This should be considered when interpreting elevated serum values.