Inhibition of Class-3 aldehyde dehydrogenase and cell growth by restored lipid peroxidation in hepatoma cell lines

Hepatoma cells have a below-normal content of polyunsaturated fatty acids; this reduces lipid peroxidation and the production of cytotoxic and cytostatic aldehydes within the cells. In proportion to the degree of deviation, hepatoma cells also show an increase in the activity of Class-3 aldehyde dehydrogenase, an enzyme important in the metabolism of lipid peroxidation products and also in that of several drugs. When hepatoma cells with different degrees of deviation were enriched with arachidonic acid and stimulated to peroxidize by ascorbate/iron sulphate, their growth rate was reduced in proportion to the quantity of aldehydes produced and to the activity of aldehyde dehydrogenase. Therefore, 7777 cells, less deviated and with low Class-3 aldehyde dehydrogenase activity, were more susceptible to lipid peroxidation products than JM2 cells. It is noteworthy that repeated treatments with prooxidant also caused a decrease in mRNA and activity of Class-3 aldehyde dehydrogenase, contributing to the decreased growth and viability. Thus, Class-3 aldehyde dehydrogenase could be considered relevant for the growth of hepatoma cells, since it defends them against cell growth inhibiting aldehydes derived from lipid peroxidation.     

PPARgamma-mediated ALDH1A3 suppression exerts anti-proliferative effects in lung cancer by inducing lipid peroxidation

Context: The metabolic function of peroxisome proliferator-activated receptor gamma (PPARγ) in lung cancer remains unclear.

Objectives: To determine the relationship of PPARγ on ALDH1A3-induced lipid peroxidation to inhibit lung cancer cell growth.

Materials and methods: In silico analysis using microarray dataset was performed to screen the positive correlation between PPARγ and all ALDH isoforms. NUBIscan software and ChIP assay were used to identify the binding sites (BSs) of PPARγ on ALDH1A3 promoter. The expression of ALDH1A3 under thiazolidinedione (TZD) treatment was evaluated by QPCR and Western Blot in HBEC and H1993 cell lines. Upon treatment of TZD, colony formation assay was used to check cell growth inhibition and 4-hydroxy-2-nonenal (4HNE) production as lipid peroxidation marker was determined by Western Blot in PPARγ positive cell H1993 and PPARγ negative cell H1299.

Results: Compared to other ALDH isoforms, ALDH1A3 showed the highest positive correlation to PPARγ expression. ALDH1A3 upregulated PPARγ expression while PPARγ activation suppressed ALDH1A3. Among 2 potential screened PPARγ response elements, BS 1 and 2 in the promoter of ALDH1A3 gene, PPARγ bound directly to BS2. Ligand activation of PPARγ suppressed mRNA and protein expression of ALDH1A3. Growth inhibition was observed in H1993 (PPARγ positive cell) treated with PPARγ activator and ALDH inhibitor compared to H1299 (PPARγ negative cell). PPARγ activation increased 4HNE which is known to be suppressed by ALDH1A3.

Conclusions: ALDH1A3 suppression could be one of PPARγ tumor suppressive function. This study provides a better understanding of the role of PPARγ in lung cancer.     

ALDH3B1 protects interfollicular epidermal cells against lipid peroxidation via the NRF2 pathway

Reactive oxygen species (ROS) production is critical for the initiation of wound repair; however, persistently high levels of ROS can lead to lipid peroxidation in cells and thus affect wound healing. Iron is a transition metal that is an essential component of almost all living cells and organisms. When present in excess in cells and tissues, iron disrupts redox homeostasis and catalyzes the generation of ROS, leading to increased lipid peroxidation. In this study, we found that after treating interfollicular epidermal (IFE) cells with different concentrations of holotransferrin (0 µg/ml, 1 µg/ml, 10 µg/ml, 100 µg/ml, and 1 mg/ml), the intracellular iron content increased, and cell viability and function did not differ significantly among the treatment groups of cells. In addition, the level of lipid peroxidation in IFE cells did not increase following holotransferrin treatment. We speculated that there is a protective mechanism within IFE cells that reduces the occurrence of intracellular lipid peroxidation. We also found that the elevated intracellular iron content of IFE cells was accompanied by elevated ALDH3B1 expression. We investigated the effect of ALDH3B1 on the level of lipid peroxidation in IFE cells and found that elevated ALDH3B1 expression decreased the damage to IFE cells induced by lipid peroxidation. In addition, the NRF2 pathway was found to affect the expression of ALDH3B1, which in turn affected lipid peroxidation in IFE cells. These findings suggest that in IFE cells, activation of the NRF2 pathway can increase the expression of ALDH3B1 and thus reduce the production of intracellular ROS and the occurrence of intracellular lipid peroxidation. Therefore, ALDH3B1 may be a potential target for the treatment of chronic wounds.     

Lysosomal lipolytic enzymes,lipid peroxidation,and injury

Summary We have used highly purified lysosomes to investigate three models of hydrolytic injury by lysosomal phospholipases. Lysosomes, enriched up to 70-fold in marker enzyme activities, can be isolated from homogenized hepatic tissue by differential centrifugation and subsequent free flow electrophoresis. These organelles remain latent and can also be utilized to obtain lysosol, the soluble fraction of the lysosomes tissue containing acid active phospholipases. The first model investigated the effect of lysosol on non-lysosomal membranes. When this soluble fraction was incubated with plasmalemma (sarcolemma) from cardiac cells, selective hydrolysis of the phospholipids was observed: phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin were the preferred substrates, and only lysophosphatidylcholine and lysophosphatidylethanolamine accumulated in significant amounts. Hydrolysis of sphingomyelin was enhanced significantly by Triton-X-100. In the second model, when intact lysosomes were incubated at acid pH, hydrolysis of phospholipids by the endogenous lipases was observed. Once again this lipolysis was specific for phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin: significant amounts of lysophospholipids also accumulated in this model. Concurrent with these lipid changes, an increase in lysosomal permeability also occurred and pH 5.0 was optimal for this lipolytic activity. However, no phospholipase activity was detected when lysosomes were incubated at pH ranges found in acidotic tissue (pH 6.0 or higher). In the third model, lysosomes were incubated at pH 6.0 in the presence of exogenously generated free radicals (dihydroxyfumarate-FeADP). A rapid loss of membrane phospholipids was observed, and most of this loss could be contributed to peroxidation of membrane phospholipids; the production of malondialdehyde preceded loss of N-acetylglucosaminidase from the lysosome. However, significant accumulation of lysophospholipids, from 2% at control time to 6.6 and 8.7% at 10 and 20 minutes, suggested that lysosomal phospholipase were hydrolyzing lysosomal phospholipids. Thus, we hypothesize that this free radical-induced lipolysis is a result of peroxidized phospholipids serving as preferred substrate for phospholipases at pH 6.0.     

Analysis of ALDH1A2, CYP26A1, CYP26B1, CRABP1, and CRABP2 in human neural tube defects suggests a possible association with alleles in ALDH1A2

BACKGROUND: Vitamin A (retinol), in the form of retinoic acid (RA), is essential for normal development of the human embryo. Studies in the mouse and zebrafish have shown that retinol is metabolized in the developing spinal cord and must be maintained in a precise balance along the anteroposterior axis. Both excess and deficiency of RA can affect morphogenesis, including failures of neural tube closure. METHODS: We chose to investigate 5 genes involved in the metabolism or synthesis of RA, ALDH1A2, CYP26A1, CYP26B1, CRABP1, and CRABP2, for their role in the development of human neural tube defects, such as spina bifida. RESULTS: An association analysis using both allelic and genotypic single-locus tests revealed a significant association between the risk for spina bifida and 3 polymorphisms in the gene ALDH1A2; however, we found no evidence of a significant multilocus association. CONCLUSIONS: These results may suggest that polymorphisms in ALDH1A2 may influence the risk for lumbosacral myelomeningocele in humans.     

Changes in growth, lipid peroxidation and some key antioxidant enzymes in chickpea genotypes under salt stress

The present study was conducted to evaluate the effect of NaCl on growth and some key antioxidants in chickpea. Eight genotypes of chickpea were grown hydroponically for 15 days and then treated with different concentrations of salt [0 mM (T0), 25 mM (T1), 50 mM (T2), 75 mM (T3), and 100 mM (T4)]. Salinity showed marked changes in growth parameters (fresh and dry weight of root and shoot). The level of lipid peroxidation was measured by estimating malondialdehyde content. Lipid peroxidation increases with the increase in NaCl concentration in all genotypes but salt-tolerant genotypes (SKUA-06 and SKUA-07) were least affected as compared to other genotypes. The chlorophyll content was also affected with elevated levels of NaCl. Increased concentration of salt increased the activity of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase in all chickpea genotypes but maximum activity was observed in salt-tolerant (SKUA-06 and SKUA-07) genotypes. Two genotypes of salt-tolerant and salt-sensitive varieties were analyzed further by real time PCR which revealed that the expression of SOD, APX and CAT genes were increased by NaCl in the salt-tolerant variety. The enhancement in tolerance against salt stress indicates that the genes involved in the antioxidative process are triggered by oxidative stress induced by environmental change. The results indicate that NaCl-induced oxidative stress hampers the normal functioning of the cell. The efficient antioxidants play a great role in mitigating the effect of NaCl stress in chickpea. This screening of NaCl-tolerant genotypes of chickpea can be performed on salt-affected land.     

Activities of antioxidant and redox enzymes in human normal hepatic and hepatoma cell lines

The cellular defense system (including glutathione, glutathione-related enzymes, antioxidant and redox enzymes) plays a crucial role in cell survival and growth in aerobic organisms. To understand its physiological role in tumor cells, the glutathione content and related enzyme activities in the human normal hepatic cell line, Chang and human hepatoma cell line, HepG2, were systematically measured and compared. Superoxide dismutase, catalase, and glutathione peroxidase activities are 2.8-, 4.3-, and 2.9-fold higher in HepG2 cells than in Chang cells. Total glutathione content is also about 1.4-fold higher in HepG2, which is supported by significant increases in gamma-glutamylcysteine synthetase and glutathione synthetase activities. Two other glutathione-related enzymes, glutathione reductase and gamma-glutamyltranspeptidase, are upregulated in HepG2 cells. However, thioredoxin reductase and glutathione S-transferase activities are significantly lower in HepG2 cells. These results propose that defense-related enzymes are largely modulated in tumor cells, which might be linked to their growth and maintenance.     

Changes in the lipid and protein components of thymus cell membrane during lipid peroxidation]

Nonenzymatic lipid peroxidation in thymus cell plasma membranes was studied. The composition of lipid and protein components, intensity of fluorescence of the membrane probes (1-anilinonaphthalene-8-sulfonate, 4-dimethylaminochalcon, eosin, pyronin and rhodamine), fluorescence polarization of tryptophan residues of membrane proteins and quenching by acrylamide of intrinsic fluorescence of proteins were determined. Induction of lipid peroxidation by the Fe(2+)-ascorbate system caused changes in the composition and structure of lipids. This was paralleled with changes in the structural-dynamic organization of membrane proteins, transition of some peripheral proteins to the water phase and increased solubilization of integral proteins by Triton X-100.     

Stemodin-derived analogues with lipid peroxidation, cyclooxygenase enzymes and human tumour cell proliferation inhibitory activities

A series of analogues, derived from the antiviral and cytotoxic diterpene stemodin, were prepared and evaluated for their lipid peroxidation (LPO), cyclooxygenase enzyme-1 (COX-1) and -2 (COX-2), and tumour cell proliferation inhibitory activities. Oxidation of stemodin produced stemodinone, which was then converted to stemod-12-en-2-one. Reaction of the latter under Petrow conditions (bromine; silver acetate/pyridine) yielded mainly dibrominated abeo-stachanes. Solvolysis of the dibromo compounds gave products of hydrolysis, some with rearranged skeleta. In the lipid peroxidation inhibitory assay three of the compounds exhibited prominent activity. Interestingly, all the analogues showed higher COX-1 enzyme inhibition than COX-2. Although a few of the diterpenes limited the growth of some human tumour cell lines, most compounds induced proliferation of such cells.     

Activities of enzymes of lipid metabolism in Morris hepatoma 7800 C1 cells

(1) The rate of palmitate oxidation in the 7800 C1 Morris hepatoma cells was about 60% of the activity observed in hepatocytes. The stimulatory effect of glucagon in hepatocytes was not observed in the hepatoma cells. The rate of fatty acid synthesis from [2-14C]acetate in the hepatoma cells was 1/20 of the activity in hepatocytes. The conversion of [2-14C]acetate to cholesterol was not different in the two kinds of cell. (2) Acetyl-CoA carboxylase and fatty acid synthetase were significantly decreased in the hepatoma cells. The hepatoma cells had, however, raised activities of malate dehydrogenase (decarboxylating), and glucose-6-phosphate and 6-phosphogluconate dehydrogenases. (3) The activities of the enzymes were not affected by different concentrations of glucose or palmitate in the culture medium. Insulin, dexamethasone, triiothyronine and glucagon had no effect on the enzyme activities. This is in contrast to the adaptation of the peroxisomal beta-oxidation system, which is induced by fatty acids and modified by hormones.     

Vitamin D enzymes (CYP27A1, CYP27B1, and CYP24A1) and receptor expression in non-melanoma skin cancer

The relationship between vitamin D metabolic enzymes (CYP27A1, CYP27B1, and CYP24A1) and vitamin D receptor (VDR) in nonmelanoma skin cancer (NMSC) development and progression is not entirely clear. However, several clinical studies and in vitro reports indicate a connection between vitamin D metabolic key players and NMSCs by demonstrating inhibitory effects on tumor cells and positive effects in skin cancer prevention of higher circulatory 25-hydroxyvitamin D [1]. Vitamin D synthesis is mediated via mitochondrial cytochrome P450 family hydroxylase enzymatic reactions: anabolic and catabolic hydroxylases (CYP27A1, CYP27B1, and CYP24A1).     

Changes in photosynthesis, antioxidant enzymes and lipid peroxidation in soybean seedlings exposed to UV-B radiation and/or Cd

Aims

With a high growth rate and biomass production, bamboos are frequently used for industrial applications and recently have proven to be useful for wastewater treatment. Bamboos are considered as Si accumulators and there is increasing evidence that silicon may alleviate abiotic stresses such as metal toxicity. The aim of this study was to investigate the extent of metal concentrations and possible correlations with Si concentrations in plants.

Methods

This study presents, for the first time, reference values for silicon (Si), copper (Cu) and zinc (Zn) concentrations in stems and leaves of various bamboo species grown under the natural pedo-climatic conditions of the island of Réunion (Indian Ocean).

Results

A broad range of silicon concentrations, from 0 (inferior to detection limit) to 183 mg g^?1 dry matter (DM), were found in stems and leaves. Mean leaf Cu and Zn concentrations were low, i.e. 5.1 mg kg^?1 DM and 15.7 mg kg^?1 DM, respectively. Silicon, Cu and Zn concentrations increased over the following gradient: stem base?<?stem tip?<?leaves. Significant differences in Si, Cu and Zn contents (except Zn in the stem) were noted between bamboo species, particularly between monopodial and sympodial bamboo species, which differ in their rhizome morphology. Sympodial bamboos accumulated more Si and Cu than monopodial bamboos, in both stems and leaves, whereas sympodial bamboos accumulated less Zn in leaves than monopodial bamboos.

Conclusions

The findings of this study suggest that a genotypic character may be responsible for Si, Cu and Zn accumulation in bamboo.     

Involvement of CYP1A1, GST, 72TP53 polymorphisms in the pathogenesis of thyroid nodules

Specific genotypes appear to be related to the development of thyroid disease. We examined whether polymorphisms of the genes CYP1A1, GSTM1, GSTT1, and TP53 at codon 72 are associated with increased risk for thyroid nodules. Blood samples were obtained from 122 thyroid patients with nodules and from 134 healthy control individuals from Goiania city, GO, Brazil. We found no significant association of CYP1A1m1 and CYP1A1m2 genotypes with thyroid diseases (P > 0.05). The null genotypes of GSTM1 and GSTT1 genes were predominant in patients with nodules, indicating that individuals that possess these genotypes have a predisposition for thyroid disease. The genotype p53Arg Arg was associated with a low risk for thyroid cancer (OR = 0.15; P < 0.0001), indicating that the arginine allele in homozygosis could have a protective effect against carcinogenesis. On the other hand, the p53ArgPro genotype was significantly associated with malignant neoplastic nodules (OR = 3.65; P = 0.001). Interindividual variation in susceptibility to thyroid diseases could provide new perspectives for early diagnosis, prognosis and treatment, indicating which patients with thyroid nodules will benefit from treatment, depending on specific polymorphic profiles.     

Characterization of CYP1A2, CYP2C19, CYP3A4 and CYP3A5 polymorphisms in South Brazilians

Potential causes of variability in drug response include intrinsic factors such as ethnicity and genetic differences in the expression of enzymes that metabolize drugs, such as those from Cytochrome P450 (CYPs) superfamily. Pharmacogenetic studies search for genetic differences between populations since relevant alleles occur with varying frequencies among different ethnic populations. The Brazilian population is one of the most heterogeneous in the world, resulting from multiethnic admixture of Amerindians, Europeans, and Africans across centuries. Since the knowledge of CYP allele frequency distributions is relevant to pharmacogenetic strategies and these data are scarce in the Brazilian population, this study aimed to describe genotype and allele distributions of 15 single nucleotide polymorphisms (SNPs) at CYP 1A2, 2C19, 3A4, and 3A5 genes in African and European descents from South Brazil. A sample of 179 healthy individuals of European and African ancestry was genotyped by the MassARRAY SNP genotyping system. CYP3A5*3, CYP1A2*1F, CYP3A4*1B, and CYP2C19*2 were the most frequent alleles found in our sample. Significant differences in genotype and allelic distribution between African and European descents were observed for CYP3A4 and CYP3A5 genes. CYP3A4*1B was observed in higher frequency in African descents (0.379) than in European descents (0.098), and European descents showed higher frequency of CYP3A5*3 (0.810) than African descents (0.523). Our results indicate that only a few polymorphisms would have impact in pharmacogenetic testing in South Brazilians. Further studies with larger sample sizes are required also among other Brazilian regions.     

Restoration of hydroperoxide-dependent lipid peroxidation by 3-methylcholanthrene induction of cytochrome P-448 in hepatoma microsomes

Microsomal membranes from the slow-growing Morris hepatoma 9618A catalyze, in the presence of t-butyl hydroperoxide, lower rates of lipid peroxidation than rat liver microsomes. The cytochrome P-450 content of hepatoma microsomes is about 40% that of the liver. SKF 525-A, an inhibitor of mixed-function oxidase, produces in hepatoma microsomes a P-450 type I binding spectrum similar to that of hepatic microsomes. The concentration of the inhibitor required for half-maximal spectral change is about 2 microM in both microsome types. SKF 525-A or ethylmorphine inhibit lipid peroxidation of normal and tumor microsomes to the same extent (about 60%). Treatment of the tumor-bearing rats with 3-methylcholanthrene increases the hepatoma cytochrome P-450 to values comparable to those of control membranes, although the hemoprotein has a peak in the CO-reduced difference absorption spectrum at 448 nm. The cytochrome P-448 induction is accompanied by an almost complete restoration of the hydroperoxide-dependent lipid peroxidation.     

Free fatty acids,lipid peroxidation,and lysosomal enzymes in experimental focal cerebral ischemia in primates: Loss of lysosomal latency by lipid peroxidation

Experimental focal cerebral ischemia was produced in monkeys (Macaca radiata) by occlusion of the right middle cerebral artery (MCA). The release of the lysosomal glycosidases, -d-hexosaminidase, -l-fucosidase and -d-mannosidase into the soluble fraction in the right basal ganglia of the experimental animals was measured at different periods from 30 min to 12 hr after occlusion and compared with the corresponding sham operated control animals. There was a significant increase in the released lysosomal enzymes in the MCA occluded animals at all periods and particularly at 4 hr after occlusion. The CSF from the experimental animals also showed elevated levels of hexosaminidase and fucosidase. The free fatty acids (FFA) measured in the basal ganglia at 30 min and 2 hr after occlusion showed a 100 fold increase in the experimental animals. The predominant fatty acid released was linoleic acid (18:2) followed by arachidonic acid (20:4). Lipid peroxidation in the basal ganglia measured by the thiobarbituric acid (TBA) reaction in the presence or absence of ascorbic acid also showed a significant increase in the experimental animals at all periods with a maximum at 30 min to 2 hr after occlusion. In order to assess whether lipid peroxidation causes damage to the lysosomes and release of the enzymes, a lysosome enriched P₂ fraction from the normal monkey basal ganglia was prepared and the effect of peroxidation studied. Maximum peroxidation in the P₂ fraction was observed in the presence of arachidonic acid, ascorbic acid and Fe²⁺. There was a good correlation between the extent of lipid peroxidation and the in vitro release of lysosomal hexosaminidase from the P₂ fraction. Anti-oxidants which strongly inhibited lipid peroxidation in the P₂ fraction prevented the release of hexosaminidase. The results suggested that in ischemia produced by MCA occlusion lipid peroxidation which damages the lysosomal membrane causes the release of lysosomal hydrolytic enzymes.Abbreviations used BHA butylated hydroxyanisole - BHT butylated hydroxytoluene - FFA free fatty acids - MCA middle cerebral artery - MDA malonaldehyde - PUFA polyunsaturated fatty acids - TBA thiobarbituric acid