Lead alters the developmental profile of the galactolipid metabolic enzymes in cultured oligodendrocyte lineage cells.

Lead is a neurotoxicant that can cause myelin deficits. Galactolipids are expressed during differentiation of oligodendrocyte lineage cells and accumulate in myelin. To examine the impact of lead on oligodendroglial differentiation, galactolipid metabolism in cultured oligodendrocyte lineage cells exposed to the metal was studied. Oligodendrocyte progenitor cells obtained from newborn rat pups were exposed to 1 microM lead acetate for 24 h prior to maintenance of the cells in medium containing the metal salt for 0, 2, or 6 days of differentiation. Lead caused approximately 50% reduction in levels of the galactolipid biosynthetic transferases, UDP-galactose:ceramide galactosyltransferase and 3'-phosphoadenosine-5'-phosphosulfate:galactocerebroside sulfotransferase, as compared to sodium-treated controls, in cultures of oligodendrocyte lineage cells following 2 days of differentiation. The activities of the galactolipid catabolic hydrolases, galactocerebroside-beta-galactosidase and arylsulfatase A, were reduced by 20%. Following 6 days of differentiation, lead-exposed cells exhibited levels of all the enzymes, except for arylsulfatase A, similar to those of the control cells. These results are consistent with the lead-induced delay of oligodendrocyte differentiation, as evidenced by the emergence of stage-specific immunochemical markers and the observed change in the developmental activity profile of 2',3'-cyclic nucleotide 3'-phosphohydrolase. The activity of arylsulfatase A in lead-treated 6-day oligodendrocytes was significantly less than that found in control cultures. This effect is consistent with the lead-induced reduction of arylsulfatase A in human fibroblasts caused by mis-sorting the newly-synthesized enzyme. The perturbation of galactolipid metabolism by lead during developmental maturation of oligodendrocytes may represent a contributing mechanism for lead-induced neurotoxicity.

Lead exposure delays the differentiation of oligodendroglial progenitors in vitro.

Lead (Pb) is an environmental neurotoxicant that can cause hypo- and demyelination. Oligodendrocytes (OLs), the myelin-forming cells in the central nervous system, may be a possible target for Pb toxicity. The present study describes the effect of Pb on the maturation of rat OL progenitor (OP) cells and the developmental expression of myelin-specific galactolipids. Dose-response studies showed that OP cultures were more sensitive to Pb than mature OLs. Pb delayed the differentiation of OL progenitors, as demonstrated by cell morphology and immunostaining with a panel of stage-specific differentiation markers. Pb given prior to and during differentiation caused a decrease in the biosynthesis of galactolipids in both undifferentiated and differentiated OLs, as detected by metabolic radiolabeling with 3H-D-galactose. While the ratios of galacto/gluco-cerebrosides, hydroxy fatty acid/nonhydroxy fatty acid galactolipids, and galactocerebrosides/sulfatides increased in control cultures during cell differentiation, Pb treatment prevented these changes. The results suggest that chronic Pb exposure may impact brain development by interfering with the timely developmental maturation of OL progenitors.

Lead causes human fibroblasts to mis-sort arylsulfatase A.

Lead exposure causes cognitive and behavioral deficits in some children. We have proposed that the effects of single nucleotide polymorphisms (SNP) of the human pseudodeficient arylsulfatase A (ARSA) gene that result in reduced levels of the enzyme, and lead concentrations that decrease ARSA activity, culminate in cellular enzymic activity that is below a critical threshold required for the normal nervous system function. Human fibroblasts grown in the presence of lead acetate exhibit a 65% decrease in ARSA protein, resulting in a significant decrease in the ability to catabolize sulfatide in cells from individuals with the SNP(s) of pseudodeficient ARSA, but not those from subjects with the normal gene (Poretz et al., Neurotoxicology 21 (2000) 379). The present study examines the potential of lead to affect the biosynthesis, trafficking and turnover of ARSA in human fibroblasts. Fibroblasts, grown in 20 microM lead, displayed a 44--58% increase in the rate of proliferation. Lead caused a decrease of approximately 33% in the accumulation of newly synthesized intracellular ARSA. This difference was not due to increased rates of intracellular degradation of ARSA or decreased levels of ARSA mRNA. Lead, however, caused the newly synthesized enzyme to be trafficked through the secretion pathway, resulting in decreased amounts of the enzyme in intracellular compartments. Though lead exposure results in increased cellular proliferation, it appears to cause decreased intracellular steady-state levels of ARSA by affecting the sorting cues and/or mechanisms directing the enzyme to lysosomes.

Lead exposure affects levels of galactolipid metabolic enzymes in the developing rat brain.

Lead poisoning is known to cause myelin defects. Galactolipids are the major lipid components of myelin and myelin-competent oligodendrocytes. The present study examines the cellular activity of enzymes involved in the galactolipid pathway, tissue concentrations of galactolipids, and the cellular activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) in rat pups exposed to lead in utero and subsequently through maternal milk from exposed mothers and in drinking water following weaning. Pups from control and lead-treated groups (500 or 2000 ppm lead in the drinking water) were euthanized by decapitation on postnatal day 7, 14, 21, 35, or 56. Lead decreased levels of galactolipids and the oligodendrocyte marker CNPase in the brain to a similar degree. The ratios of galactocerebrosides/sulfatides and nonhydroxy fatty acid/hydroxy fatty acid forms of the galactolipids were not altered by lead treatment. In contrast, the activities of the galactolipid metabolic enzymes were reduced to a degree significantly greater than that of CNPase or galactolipids. These results are consistent with previously obtained data indicating that in vitro cultured oligodendroglial progenitor cells are a target for Pb toxicity. Chronic Pb exposure may impact on brain development by impairing timely myelin production due to perturbation of the early developmental commitment of oligodendroglial progenitors. It is further suggested that perturbation of the galactolipid pathway during the developmental maturation of oligodendrocytes may represent a contributing mechanism for Pb-induced neurotoxicity.

The interaction of lead exposure and arylsulfatase A genotype affects sulfatide catabolism in human fibroblasts.

Lead exposure causes cognitive and behavioral deficits in some affected children. We propose that a contributing mechanism for the neurological damage is that lead induces critically low levels of arylsulfatase A (ASA) at sensitive stages of nervous system development. It is hypothesized that the combined effects of a single nucleotide polymorphism (SNP) in human ASA which results in reduced levels of the enzyme, and lead concentrations which decrease ASA activity culminate in cellular enzymic activity that is below a critical threshold required for the maintenance of normal nervous system function. Human fibroblasts grown in the presence of 20 microM lead acetate exhibit a more than 60% decrease of cellular ASA enzyme protein. Lead treatment of cells from individuals with the SNP(s) of pseudodeficient ASA, but not those from subjects with the normal gene, results in a significant decrease in ability of the cells to desulfate sulfatide, the substrate of ASA. The decrease in the degree of sulfatide catabolism is consistent with possible enhanced lead-induced neurobehavioral effects in individuals homozygous for the pseudodeficiency polymorphism(s) of ASA.

Sites of photodamage induced by photodynamic therapy with a chlorin e6 triacetoxymethyl ester (CAME).

The acetoxymethyl ester of chlorin e6 (CAME) was initially designed to be a hydrophobic photosensitizing agent that would be recognized by an endocytic pathway and initially accumulated in lysosomes. This was expected to lead to hydrolysis of the ester groups, followed by redistribution of the free chlorin to other subcellular sites. In this study, we examined the patterns of localization of CAME and of subsequent photodamage in murine leukemia L1210 cells. The drug was initially localized at intracellular sites, yielding a pattern similar to that obtained with a fluorescent probe for acidic intracellular vesicles and endosomes. A brief (30 min) incubation with 10 microM CAME followed by irradiation led to mitochondrial photodamage and apoptotic cell death. At a higher drug level, or with a longer incubation time, we observed additional photodamage to the plasma membrane and to lysosomes. The higher photodynamic therapy dose led to inhibition of apoptosis, with cell death likely occurring via a necrotic process. Distribution of CAME among the components of human plasma was to albumin > high-density lipoprotein > low-density lipoprotein. These results have implications concerning the likely mechanism of CAME accumulation and subcellular distribution.

Effect of dietary chlorophyll derivatives on mutagenesis and tumor cell growth.

Much attention in recent years has been given to the antigenotoxicity of chlorophyll. Chlorophyll, however, is known to be converted into pheophytin, pyropheophytin, and pheophorbide in processed vegetable food and following ingestion by humans. Studies were conducted on the antimutagenic and tumoricidal potencies of these compounds. All the chlorophyll derivatives tested exhibit identical antimutagenic effect towards 3-methylcholanthrene (3-MC), suggesting that the porphyrin nucleus may complex directly with the mutagen. It does not exclude, however, another mechanism of activity involving inactivation the enzymatic transformation of 3-MC. In contrast, the action of N'-nitro-N'-nitrosoguanidine (MNNG) depends upon structural differences between the chlorophyll derivatives. It is significantly lower when the phytol-containing pheophytin and pyropheophytin are tested as to that of the phytol-lacking pheophorbide. The higher concentrations of the chlorophyll derivatives were required to reduce the mutagenicity of MNNG than needed for 3-MC. The cytotoxicity of chlorophyll derivatives against tumor cells also was evaluated. The cellular uptake and inhibition of myeloma cell multiplicity were found to be greater for pheophorbide than for pheophytin. Calculated on the amount of cell associated chlorophyll derivative, however, pheophytin was more cytostatic/cytotoxic than pheophorbide. The results presented in this report indicate that food sources that yield chlorophyll derivatives may play a significant role in cancer prevention. Teratogenesis Carcinog. Mutagen. 19:313-322, 1999.

The R496H mutation of arylsulfatase A does not cause metachromatic leukodystrophy.

Deficiency of arylsulfatase A (ARSA) enzyme activity causes metachromatic leukodystrophy (MLD). A number of ARSA gene mutations responsible for MLD have been identified. Recently, the R496H mutation of ARSA was proposed to be a cause of MLD (Draghia et al., 1997). We have investigated the R496H mutation and found this mutation at a relatively high frequency in an African American population (f=0.09, n=61 subjects). The ARSA enzyme activity in subjects with and without the R496H mutation was determined and found to be normal. It is therefore concluded that the R496H mutation of ARSA does not negatively influence the activity of ARSA and is not a cause of MLD.

An acidic modification of the cytoplasmic domain contributes to the charge heterogeneity of the MHC class I antigens.

Polypeptide phosphorylation and sialylation of the glycan moieties contribute to the charge heterogeneity of the class I major histocompatibility complex glycoproteins. The present study demonstrates that a unique acidic modification unrelated to phosphorylation or glycosylation also affects the charge heterogeneity of the H2-Kk heavy chain of BW5147 lymphoma cells. In vitro cultivation of BW5147 cells results in changes in charge heterogeneity of the H2-Kk heavy chains due to the unique acidic modification. Sequential papain digestion of the 45 000 Mr H2-Kk glycoprotein yields a 42 500 Mr glycopolypeptide initially, followed by production of a 39 000 Mr glycopolypeptide. Results from experiments designed to localize and characterize the novel acidic modification suggest that the modification resides in the segment of the H2-Kk polypeptide located between the two papain cleavage sites. This portion of the polypeptide consists of the transmembrane region and part of the cytoplasmic domain of the H2-Kk heavy chain. At steady state, 25% of the total cell surface H2-Kk possesses this modification. In addition, the modification is mutually exclusive with the phosphorylation of the H2-Kk heavy chain at Ser-333. The possible biological significance of the novel modification of class I antigens is discussed.

Antimutagenicity, cytotoxicity and composition of chlorophyllin copper complex.

The preparation of chlorophyllin copper complex (CCC), shown to be a tumor promoter in an animal model (Nelson, R.L. (1992) Chlorophyllin, an antimutagen, acts as a tumor promoter in the rat-dimethylhydrazine colon carcinogenesis model. Anticancer Res., 12, 737-740), also inhibits the activities of direct- and indirect-acting mutagens in the Salmonella assay and exhibits cytostatic and cytocidal effects toward myeloma cells. Data from elemental analyses, spectrophotometry and reversed-phase high-performance liquid chromatography indicate that CCC preparations generally used in antimutagenic/anticarcinogenic experiments are variable, complex mixtures of structurally distinct porphyrins lacking copper in some instances. This variability of the composition may be a cause for the differences reported for the tumor promotion activity of CCC.

A novel arylsulfatase A protein variant and genotype in two patients with major depression.

A new, 'diffuse, multiple banding', electrophoretic variant of arylsulfatase A protein was found in two patients with major depression. Protein analyses showed that this variant and the normal enzyme differed in amino acid sequence and/or post-translational modifications unrelated to phosphate groups and oligomannose glycans. Analysis of the arylsulfatase A genes from a subject with the new variant identified three mutations; one gene had the two mutations associated with arylsulfatase A pseudodeficiency, and the other had a G to T transversion which changes a tryptophan to cysteine in the protein. These mutations result in an arylsulfatase A protein heteromer with diffuse electrophoretic banding. The possible association of these mutations with major depression is discussed.

Association of alcoholism with the N-glycosylation polymorphism of pseudodeficient human arylsulfatase A.

The IIIa and IIIb electrophoretic variants of arylsulfatase A (EC 3.1.6.8) are 12 times more prevalent in alcoholic than in nonalcoholic populations. These variant enzymes, found in a subset of alcoholics, possess the pseudodeficient Asn350-Ser mutation of arylsulfatase A and, consequently, lack an N-linked glycan unit. These genetically determined variants of arylsulfatase A show reduced intracellular half-life, and cells from such individuals possess reduced enzymic activity. We propose that this polymorphism is an underlying genetic and biochemical factor contributing to the neuropathology and/or addiction pathway of this disease.

Structural characterization of variant forms of arylsulfatase A that associate with alcoholism.

Several electrophoretic forms of human platelet arylsulfatase A (ASA), including variant type IIIa and normal type IV(a), have been identified by nondenaturing polyacrylamide gel electrophoresis. An alcoholic population that we have analyzed is enriched in variant type IIIa compared with nonalcoholic psychiatric and normal controls. Individuals with the IIIa enzyme possess greatly reduced levels of ASA activity. To understand further the structural basis for the differences and their potential biological consequences, the nature of the ASA variant expressed by fibroblasts from different individuals was explored. The electrophoretic patterns of fibroblast ASA from the IIIa and IV(a) individuals differ in degree of phosphorylation. Furthermore, fibroblast ASA from IIIa individuals lacks an N-linked glycan found in ASA from IV(a) individuals. In addition, differences in peptide and/or posttranslational modification unrelated to the N-linked carbohydrate or phosphorylation exist between the fibroblast ASA from IIIa and IV(a) individuals. The finding that both fibroblasts and platelets exhibit related electrophoretic isoform patterns characteristic of the donor's ASA type allows for the use of fibroblasts to study the impact of ethanol on the metabolism of cells possessing different ASA types.

Arylsulfatase A: relationship of genotype to variant electrophoretic properties.

Previous work has shown that specific electrophoretic variants of arylsulfatase A occur more frequently among alcoholic patients than among psychiatric and normal controls. The present study sequenced the gene for two of these electrophoretic variants, IIIa and IIIb. Both contain an A-to-G transition corresponding to substitution of Asn350 by Ser, with the resulting loss of an N-glycosylation site. The difference in electrophoretic mobility of their gene products is due to a mutation in the IIIb gene resulting in the replacement of Arg496 by His. Evidence is presented that individuals possessing either of two other electrophoretic variants, Va and Vb, are heterozygous for a normal ASA allele and either a IIIa or IIIb allele, respectively. Thus, the relationship between the phenotype of the electrophoretic banding patterns, IIIa, IIIb, Va, and Vb, and their corresponding genotypes has been elucidated.

In-vitro photocytotoxicity of lysosomotropic immunoliposomes containing pheophorbide a with human bladder carcinoma cells.

Pheophorbide a is a photocytotoxic agent. To develop a tissue-specific, intracellularly targeted photoactive system, pheophorbide a was incorporated into immunoliposomes coated with a monoclonal antibody (T-43) directed against the T-24 bladder tumor cell line. The efficacy of this system was studied in vitro using the human bladder tumor cell line MGH-U1. Uptake and localization were determined by the fluorescence of the immunoliposome markers within biochemically resolved subcellular components. The results demonstrate localization of the immunoliposome markers within the lysosomes of the tumor cells. Specific monoclonal antibody enhancement of the immunoliposomes uptake by MGH-U1 cells was demonstrated by the use of soluble T-43 monoclonal antibody as a competitive inhibitor. Pheophorbide-a-loaded immunoliposomes were shown to be photocytotoxic towards MGH-U1 cells at concentrations equivalent to photosensitizer at 500 ng ml-1. Treated cells, when protected from light, showed no cytotoxicity. These results demonstrate that uptake of pheophorbide-a-containing immunoliposomes by target cells and subsequent delivery to the lysosomes cause photoactivated killing of tumor cells. The utilization of immunoliposomes for intracellular lysosomal targeting of photoactive drugs to tumor cells constitutes a potentially valuable approach to photodynamic therapeutics.

Metabolically convertible lipophilic derivatives of pH-sensitive amphipathic photosensitizers.

We propose the use of acetoxymethyl esters of pH-sensitive amphipathic photosensitizers (PS) for photodynamic therapy (PDT). These compounds may be applicable for PDT involving endocytosis of lipophilic carriers leading to lysosomal uptake of the esterified PS by target cells. Partial and/or total enzymatic de-esterification may result in the extralysosomal distribution of the photoactive agents, possibly culminating in a multisite photochemical response. We report here the synthesis and properties of chlorin e6 triacetoxymethyl ester (CAME) and pheophorbide a acetoxymethyl ester (PAME). Chlorin e6 and pheophorbide a are photocytotoxic chlorins that possess free carboxylate groups and exhibit optimum wavelengths of excitation substantially red shifted relative to hematoporphyrin derivative. Acetoxymethyl esterification of chlorin e6 and pheophorbide a was accomplished with bromomethyl acetate. High-performance liquid chromatography allowed for the purification of PAME, in 87% purity, and CAME, in 63% yield and 94% purity, as well as the detection of the presumed mono- and diesters of chlorin e6 as transient intermediates in the synthesis of CAME. The ultraviolet-visible absorption, fluorescence excitation and emission, NMR and mass spectra of the chlorin e6 triester are consistent with those expected for CAME. The pH-sensitive amphipathicity of pheophorbide a and chlorin e6 but not CAME was demonstrated using a water/1-octanol partition assay. The production of pheophorbide a from PAME and the sequential formation of the di- and monoesters and free chlorin e6 from CAME, by the action of lysosomal esterases obtained from cancer cells, demonstrate the potential of cellular enzymes to convert the lipophilic esters to pH-sensitive amphipathic PS.(ABSTRACT TRUNCATED AT 250 WORDS)

Two-dimensional poly(acrylamide) electrophoresis of fluoresceinated glycopeptides. Resolution and structural characterization of ovalbumin glycans.

The microheterogeneous mixture of fluoresceinated glycopeptides (FGPs) obtained from the single site of glycosylation of chicken ovalbumin was resolved by a combination of discontinuous electrophoresis in a high-density poly(acrylamide) gel (PAGE) for sizing, in conjunction with borate-PAGE. Two FGPs of similar size but with different mobilities in borate-PAGE were purified and characterized by sequential exoglycosidase digestion and sizing on the discontinuous PAGE system, as well as by methylation analysis. The two FGPs of identical size are distinct and have structures beta-D-Glc pNAc-(1-->2)-alpha-D-Man p-(1-->3)-[beta-D-Glc pNAc-(1-->4)]-[beta-D-Glc pNAc-(1-->2)-alpha-D- Man p-(1-->6)]-beta-D-Man-p-(1-->4)-beta-D-Glc pNAc-(1-->4)-beta-D-Glc pNAc-1-->R and alpha-D-Man p-(1-->2)-alpha-D-Man p-(1-->3 or 6)-[alpha-D-Man p-(1-->3)-[alpha-D-Man p-(1-->6)]-alpha-D-Man p-(1-->6 or 3)]-beta-D-Man p-(1-->4)-beta-D-Glc pNAc-(1-->4)-beta-D-Glc pNAc-1-->R (R = Asn-(amino acids)-fluorescein). The results demonstrate that two-dimensional PAGE is applicable to the separation and characterization of complex mixtures of FGPs. The procedure is rapid, sensitive, and convenient for glycopeptide mapping, and for the purification and structural characterization of glycans. Furthermore, the FGPs can be characterized with affinity matrices, such as lectins, and by methylation analysis.

The structural basis for the electrophoretic isoforms of normal and variant human platelet arylsulphatase A.

Human platelet arylsulphatase A (ASA) exhibits a multiple banding pattern when examined by PAGE. The isoform pattern (IVa) of the enzyme obtained from normal subjects differs from variants (IIIa, IIIb, IVb) which are primarily found in alcoholic patients. Alkaline phosphatase and endo-N-acetylglucosaminidase H treatments, as well as ion-exchange chromatography, demonstrate that the isoforms of ASA arise because of charge heterogeneity caused by phosphoglycan moieties. The isoform with the slowest mobility in PAGE lacks phosphate substituents. Based upon mannose-6-phosphate-receptor affinity chromatography it can be concluded that most, if not all, of the enzyme-linked phosphate is in the form of 6-O-phospho-D-mannosyl units. Lectin affinity chromatography and peptide-N-glycosidase F treatment followed by SDS/PAGE and Western-blot analysis indicate that normal platelet ASA contains two oligomannose and/or hybrid glycan moieties of which one, or both, possess a 6-O-L-fucosyl substituent on the asparagine-linked N-acetylglucosamine residue. Comparative analysis indicates that the variant IIIa and IIIb types of ASA differ from the IVa ASA with regard to the level of glycan phosphorylation and glycan structure, as well as the polypeptide structure.

Pheophorbide a-induced photo-oxidation of cytochrome c: implication for photodynamic therapy.

Pheophorbide a-induced photo-oxidation, in vitro, of cytochrome c oxidase and cytochrome c results in irreversible modifications to both protein components. Photo-oxidation of cytochrome c, as exhibited by change in its heme oxidation state, displays exponential kinetics and is detected with a lag period. Both the photo-induced inactivation of the enzyme, and destruction of the substrate ability of cytochrome c occur as complex multi-process events. Under similar experimental conditions, the loss of the substrate capability of cytochrome c develops approximately three times faster than inactivation of the enzyme. The slight lag in the photo-oxidation of cytochrome c is due to pheophorbide a-induced superoxide production. However, the relative amount of photo-oxidant produced is considerably more effective than the cytochrome c reducing capacity of the superoxide. Neither hydroxyl radical nor hydrogen peroxide are involved in the photo-oxidation of the heme function. The possibilities of heme oxidation by a singlet oxygen mediated pathway or direct electron abstraction involving the heme or apoprotein are not excluded. It is proposed that a multi-site oxidation of numerous reduced energy cofactors within cells may augment collateral enzyme inactivation in maximizing photosensitizer-induced cytotoxicity. Accordingly, amphipathic photosensitizers, capable of accessing both lipid and aqueous compartments containing reduced cofactors, may be more effective agents for photodynamic therapy than those which exhibit a high specificity of subcellular localization.