Inhibitory effects of Aphanizomenon flos-aquae constituents on human UDP-glucose dehydrogenase activity

Objective: The purpose of this study was to investigate the in vitro inhibitory effects of the edible microalga Aphanizomenon flos-aquae (AFA) on human UDP-α-d-glucose 6-dehydrogenase (UGDH) activity, a cytosolic enzyme involved both in tumor progression and in phytochemical bioavailability.

Methods: Both the hydrophilic and ethanolic AFA extracts as well as the constitutive active principles phycocyanin (PC), phycocyanobilin (PCB) and mycosporine-like amino acids (MAAs) were tested.

Results: Among AFA components, PCB presented the strongest inhibitory effect on UGDH activity, acting as a competitive inhibitor with respect to UDP-glucose and a non-competitive inhibitor with respect to NAD⁺. In preliminary experiments, AFA PCB was also effective in reducing the colony formation capacity of PC-3 prostate cancer cells and FTC-133 thyroid cancer cells.

Conclusions: Overall, these findings confirmed that AFA and its active principles are natural compounds with high biological activity. Further studies evaluating the effects of AFA PCB in reducing tumor cell growth and phytochemical glucuronidation are encouraged.     

Variation von PCB-Gemischen in Eiern und V?geln des Wattenmeeres

Zusammenfassung Bei Flußseeschwalbe und Austernfischer wurde der Einfluß von Standort und Jahr auf die Zusammensetzung der PCB-Gemische im Ei untersucht. Die Gesamtkonzentration, der Metabolisierungsgrad und die Ringstabilitätszahlen des Gemisches von 45 bestimmten PCB-Kongeneren wurden erfaßt. In den Mündungsbereichen von Elbe, Weser und in der Inneren Deutschen Bucht traten nicht nur die höchsten PCB-Konzentrationen auf, sondern auch die am weitesten metabolisierten Gemische. 1987–1989 zeigte sich im Metabolisierungsgrad bei beiden Vogelarten ein linearer Anstieg. Flußseeschwalbeneier enthielten zwar deutlich höhere PCB-Konzentrationen als Austernfischereier, doch war das Gemisch geringer metabolisiert. Deutliche Artunterschiede traten auch in den Ringstabilitätszahlen auf. Zur Beurteilung der PCB-Belastung sollte deshalb in einem Monitoring-Programm der Metabolisationsgrad auch mitberücksichtigt werden.

---

Variation of PCB mixtures in eggs of birds of the Wadden Sea

Summary The influence of location and year on the composition of PCB mixtures found in eggs was examined in the two wadden sea bird species Common Tern (Sterna hirundo) and Oystercatcher (Haematopus ostralegus). By means of 45 PCB congeners the total concentration, the degree of metabolism and the ring stability values were analysed. The highest PCB concentrations as well as the highest degree of metabolism were found in the estuaries of the rivers Elbe and Weser and in the Deutsche Bucht. The degree of metabolism of both species increased linearly from 1987 to 1989. The total PCB concentration in the eggs of Common Terns were clearly higher than those found in the eggs of Oystercatchers but the mixture was less metabolised. Furthermore, there are significant differences of the ring stability values between the two species. To assess the PCB incrimination the degree of metabolism should be considered as well in a monitoring program.

     

CAPACITY AND MODE OF PCB METABOLISM IN SMALL CETACEANS1

Based on a comparative approach using PCB isomer and congener compositions in higher animals and their food organisms, the capacity and mode of PCB metabolism in small cetaceans were studied and the following conclusions were drawn: (1) Small cetaceans can metabolize some of the lower chlorinated biphenyls and this capacity seems to be the same in all species of these animals. (2) The values of MI, an index to evaluate the capacity of PCB metabolism, showed that the metabolic capacity of small cetaceans was extremely low as compared to those of birds and terrestrial mammals. (3) The structural requirements for PCB metabolism were different in animal species, in that small cetaceans have no capacity to metabolize a group of PCBs with adjacent non-chlorinated meta and para carbons in biphenyl rings. (4) No development of PB (phenobarbital)-type enzymes, and a lower activity of MC (3-methylcholanthrene)-type enzymes were suggested in small cetaceans, which implies long-term accumulation and possible reproductive toxicity of persistent organochlorines in these animals. The present approach should provide an important insight into the physiological responses of small cetaceans to persistent toxic chemicals.     

Biodegradation and evaporation of polychlorinated biphenyls (PCBs) in liquid media

During microbial degradation of PCBs in a liquid medium, two processes influence the PCB concentration in the medium simultaneously: biodegradation and evaporation. The physical loss of PCB due to evaporation frequently causes false positive results in biodegradation experiments. Therefore, if only PCBs are monitored, the determination of the PCB concentration in both liquid and gaseous phases is necessary for a correct appraisal of biodegradation. The kinetics of PCB evaporation and biodegradation were monitored and described by a simple mathematical model. The evaporation and biodegradation rate constants for individual PCB congeners were determined for PCB degradation in liquid medium byPseudomonas stutzeri andAlcaligenes xylosoxidans, both isolated from a longterm PCB-contaminated soil.Symbols a ₁,b ₁,a ₂,b ₂ fitting parameters - c ₀ initial concentration of PCB congener in liquid medium - c _l concentration of PCB congener in liquid medium - c _ev concentration of PCB congener in sorbent - k _ev rate constant of PCB congener evaporation - k _met rate constant of PCB congener metabolization - n _s amount of PCB congener in sorbent - t _1/2 half-time of evaporation - V _t volume of liquid medium     

Polychlorinated Biphenyl/Biphenyl Degrading Gene Clusters in Rhodococcus sp. K37, HA99, and TA431 Are Different from Well-Known bph Gene Clusters of Rhodococci

Four kinds of polychlorinated biphenyl (PCB)-degrading Rhodococcus sp. (TA421, TA431, HA99, and K37) have been isolated from termite ecosystem and under alkaline condition. The bph gene cluster involved in the degradation of PCB/biphenyl has been analyzed in strain TA421. This gene cluster was highly homologous to bph gene clusters in R. globerulus P6 and Rhodococcus sp. RHA1. In this study, we cloned and analyzed the bph gene cluster essential to PCB/biphenyl degradation from R. rhodochrous K37. The order of the genes and the sequence were different in K37 than in P6, RHA1, and TA421. The bphC8 _K37 gene was more homologous to the meta-cleavage enzyme involved in phenanthrene metabolism than bphC genes involved in biphenyl metabolism. Two other Rhodococcus strains (HA99 and TA431) had PCB/biphenyl degradation gene clusters similar to that in K37. These findings suggest that these bph gene clusters evolved separately from the well-known bph gene clusters of PCB/biphenyl degraders.     

Metabolism of polychlorinated biphenyls by Solanum nigrum hairy root clone SNC-9O and analysis of transformation products

The study investigates aspects of PCB metabolism by a hairy root culture of Solanum nigrum L. (clone SNC-9O) in vitro. Standard conditions were established for efficient, up to 72% PCB conversion (22 individual PCB congeners examined in commercial mixture Delor 103, 5 g fresh biomass in 100 ml media shaken with 5 mg PCB for 14 days). The conversion products formed from three monochlorobiphenyls were monohydroxychlorobiphenyls and dihydroxychlorobiphenyls, while six dichlorobiphenyls yielded different monohydroxydichlorobiphenyls. Efficiency of the transformation of individual PCB congeners was evaluated together with phytotoxic effect on the clone SNC-9O. Major metabolites of monochlorobiphenyls analysed after extraction from biomass were hydroxylated at the position 4, and 4′, respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

Bioaugmentation with Immobilized Microorganisms to Enhance Phytoremediation of PCB-Contaminated Soil

The aim of this study was evaluate the effect of bioaugmentation by free and immobilized strains of microbial consortium on the phytoremediation of polychlorinated biphenyl (PCB)-contaminated soil using the Avena sativa, Brachiaria decumbens, Brassica juncea, and Medicago sativa plants. Alginate and biochar were used as carrier materials and free cells were used as the control. PCBs 44, 66, 118, 138, 153, 170, and 180 were chosen as indicator PCB congeners. After 60 days of plant growth, the concentration of each congener and the survival of the microbial inoculum were evaluated. The removal of the PCB congener was greater in B. juncea planted treatments and using biochar as a carrier material. PCB 66 was the congener with the highest removal percentage in all using biochar and alginate-immobilized microorganisms and free microorganisms, while PCB 170 had the lowest removal percentage in all treatments. The largest removal percentage for all congeners was obtained using biochar as a carrier material (7.2–30.3%) and the lowest with planted treatments using free microorganisms (2.3–6.8%). Real-time polymerase chain reaction (PCR) showed that the microbial inoculum survived when it was immobilized using both alginate and biochar without any significant differences between treatments; however, PCB removal percentages were obtained with biochar, which demonstrated that this carrier material has a positive effect on microbial activity.     

Genotoxic effects of PCB 52 and PCB 77 on cultured human peripheral lymphocytes

Polychlorinated biphenyls (PCBs) are known to be carcinogenic, but the mechanisms of this action are uncertain. Most, but not all, studies have concluded that PCBs are not directly mutagenic, and that much if not all of the carcinogenic activity resides in the fraction of the PCB mixture that contains congeners with dioxin-like activity. The present study was designed to determine genotoxic effects of an ortho-substituted, non-coplanar congener, 2,2′,5,5′-tetrachlorobiphenyl (PCB 52), and a non-ortho-substituted coplanar congener with dioxin-like activity, 3,3′,4,4′-tetrachlorobiphenyl (PCB 77) on cultured human peripheral lymphocytes. DNA damage was assessed by use of the comet assay (alkaline single-cell gel electrophoresis). After cell cultures were prepared, test groups were treated with different concentrations of PCB 52 (0.2 and 1 μM) and PCB 77 (1 and 10 μM) for 1 h at 37 °C in a humidified carbon dioxide incubator, and compared to a DMSO vehicle control group. The cells were visually classified into four categories on the basis of extent of migration such as undamaged (UD), low damage (LD), moderate damage (MD) and high damage (HD). The highest concentration of PCBs 52 and 77 significantly increased DNA breakage in human lymphocytes (p < 0.001). Our results indicate that both the non-coplanar PCB 52 and coplanar PCB 77 cause DNA damage, and that the ortho-substituted congener was significantly more potent than the dioxin-like coplanar congener.     

Effect of Feeding Xenobiotics on Serum High Density Lipoprotein and Apolipoprotein A-I in Rats

The effects on serum cholesterol level were examined in rats fed on various xenobiotics. The hypercholesterolemia induced by polychlorinated biphenyls (PCB) was characterized in rats, from which lipoproteins were isolated by ultracentrifugation. A dietary addition of 0.03% PCB, 0.3% chloretone, 0.1% aminopyrine, or 0.2% 2,6-di-tert-butyl-p-cresol (BHT) resulted in a significant increase in serum cholesterol, although the chemical structure of each of these xenobiotics was different. The serum cholesterol level was markedly increased by one month of PCB feeding, the effect of PCB on the serum phospholipid level being similar. The serum triglyceride level transiently increased within 7 days of feeding with PCB diet. PCB feeding resulted in the elevation of all lipoproteins, including VLDL, LDL, HDL₁, and HDL₂, a marked increase being observed in HDI₁. Both HDL₁ and HDL₂ isolated from PCB-treated rats contained more apolipoprotein A-I (apo A-I) and less apo E than normal. VLDL isolated from PCB-treated rats had more cholesterol and apo E, but less apo C than that of the control animals. These data demonstrate that PCB feeding resulted in increased VLDL rich in cholesterol and apo E, and increased HDL rich in apo A-I. This experimentally induced hypercholesterolemia resulting in apo A-I-rich HDL would be a useful model for investigating the metabolism of apo-A-I and HDL.     

Phytoremediation of soil contaminated with PCBs using different plants and their associated microbial communities

In this work, we evaluate the abilities of the plants Brassica juncea, Avena sativa, Brachiaria decumbens, and Medicago sativa to uptake polychlorinated biphenyls (PCBs) and induce degradation of soil microorganisms from contaminated soil. Removal of PCBs 44, 66, 118, 153, 170, and 180 was evaluated in both rhizospheric and nonrhizospheric soils. Microbial and bphA1 gene quantifications were performed by real-time PCR. The PCB concentrations in plant tissues and soil were determined, and a fluorescein diacetate (FDA) hydrolysis assay was used to measure microbial activity in soil. The removal percentages for all PCB congeners in planted soil versus unplanted control soil were statistically significant and varied between 45% and 63%. PCBs 118, 153, 138, and 170 were detected in Brachiaria decumbens roots at different concentrations. In planted soil, an increase in the concentration of bacteria was observed compared to the initial concentration and the concentration in unplanted control soil; however, no significant differences were identified between plants. The number of copies of the bphA1 gene was higher in rhizospheric versus non- rhizospheric soil for all plants at the end of the experiment. However, alfalfa and oat rhizospheric soil showed significant differences in the copy number of the bphA1 gene. In general, the concentration of fluorescein in the rhizospheric soil was greater than that in the nonrhizospheric soil. Although the plants had a positive effect on PCB removal, this effect varied depending on the type of PCB, the plant, and the soil.     

Effects of inhibitors on 3-O-methylglycose transport in rabbit ileum

Summary Previous studies (Goldner, Schultz & Curran,J. Gen. Physiol. 1969,53:362) have suggested a direct coupling between influxes of sugars and Na across the brush border membrane of rabbit ileum. Effects of several inhibitors, ouabain, cyanide, dinitrophenol and iodoacetate on 3-O-methylglucose fluxes were examined in an effort to obtain information about coupling of sugar transport to metabolism. The inhibitors virtually abolished net active sugar transport across the whole tissue but had less striking effects on sugar influx across the brush border membrane, particularly when the cells were prevented from gaining Na as a result of inhibitor action. However, substantial but incomplete inhibition of influx was observed when the cells were permitted to gain Na. Mucosal strips incubated with ouabain to elevate cellular Na extruded sugar against a concentration gradient when cell Na concentration exceeded that in the medium. Conversely, a small extrusion of Na from ouabain-poisoned cells was observed in the presence of an outwardly directed concentration gradient for sugar. These results provide further evidence of coupling between Na and sugar movement. Additional direct coupling of sugar movement to metabolism cannot be ruled out.     

Degradation of polychlorinated biphenyl (PCB) by a consortium obtained from a contaminated soil composed of Brevibacterium,Pandoraea and Ochrobactrum

An indigenous polychlorinated biphenyl (PCB)-degrading bacterial consortium was obtained from soils contaminated by transformer oil with a high content of PCBs. The PCB degrader strains were isolated and identified as Brevibacterium antarcticum, Pandoraea pnomenusa, and Ochrobactrum intermedium by 16S rRNA gene sequence phylogenetic analysis. The PCB-degrading ability of the consortium and of individual strains was determined by using GC/MS. The PCB-degrading capacities of the consortium were evaluated for three concentrations of transfomer oil ranging from 55 to 152 μM supplemented with 0.001% biphenyl and 0.1% of Tween 80 surfactant. PCB biodegradation by the consortium was favored in the presence of both additives and the greatest extent of biodegradation (67.5%) was obtained at a PCB concentration of 55 μM. Each bacterial species exhibited a particular pattern of degradation relating to specific PCB congeners. Isolated strains showed a moderate degradation capability towards tetra-, hepta-, and octa-chlorobiphenyls; although no effect on penta-, hexa-, and nona-chlorobiphenyls was observed. Recently, PCB degradation capacity was recognized in a Pandorea member; however, this is the first study that describes the ability of Brevibacterium and Ochrobactrum species to degrade PCBs.     

Growth characteristics of the cyanobacterium Nostoc flagelliforme in photoautotrophic, mixotrophic and heterotrophic cultivation

Nostoc flagelliforme is a terrestrial cyanobacterium with high economic value. Dissociated cells separated from a natural colony of N. flagelliforme were cultivated for 7 days under either phototrophic, mixotrophic or heterotrophic culture conditions. The highest biomass, 1.67 g L⁻¹ cell concentration, was obtained under mixotrophic culture, representing 4.98 and 2.28 times the biomass obtained in phototrophic and heterotrophic cultures, respectively. The biomass in mixotrophic culture was not the sum as that in photoautotrophic and heterotrophic cultures. During the first 4 days of culture, the cell concentration in mixotrophic culture was lower than the sum of those in photoautotrophic and heterotrophic cultures. However, from the 5th day, the cell concentration in mixotrophic culture surpassed the sum of those obtained from the other two trophic modes. Although the inhibitor of photosynthetic electron transport DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] efficiently inhibited autotrophic growth of N. flagelliforme cells, under mixotrophic culture they could grow by using glucose. The addition of glucose changed the response of N.flagelliforme cells to light. The maximal photosynthetic rate, dark respiration rate and light compensation point in mixotrophic culture were higher than those in photoautotrophic cultures. These results suggest that photoautotrophic (photosynthesis) and heterotrophic (oxidative metabolism of glucose) growth interact in mixotrophic growth of N. flagelliforme cells.     

Molecular genetics and evolutionary relationship of PCB-degrading bacteria

Biphenyl-utilizing soil bacteria are ubiquitously distributed in the natural environment. They cometabolize a variety of polychlorinated biphenyl (PCB) congeners to chlorobenzoic acids through a 2,3-dioxygenase pathway, or alternatively through a 3,4-dioxygenase system. Thebph genes coding for the metabolism of biphenyl have been cloned from several pseudomonads. The biochemistry and molecular genetics of PCB degradation are reviewed and discussed from the viewpoint of an evolutionary relationship.Abbreviations BP biphenyl - bph BP/PCB-degradative gene - 23DHBP 2,3-dihydroxybiphenyl - HPDA 2-hydroxy-6-oxo-6-phenylhexa 2,4-dienoic acid - KF707 P. pseudoalcaligenes strain KF707 - LB400 Pseudomonas sp. strain LB400 - PCB polychlorinated biphenyls - Q1 P. paucimobilis strain Q1tod; toluene catabolic gene     

Effect of chlorobenzoates on the degradation of polychlorinated biphenyls (PCB) by Pseudomonas stutzeri

Chlorobenzoic acids (CBA) are frequently dead-end products of partial aerobic biodegradation of polychlorinated biphenyls (PCB). When CBA produced from PCB accumulate in the growth medium, they can inhibit the bacterial growth and consequently, slow down PCB biodegradation. In this study, the effects of seven mono- and dichlorinated CBA on growth of Pseudomonas stutzeri on different substrates and on the PCB degradation by this strain in a liquid mineral medium were tested. 3-CBA was the strongest growth inhibitor for P. stutzeri growing on glucose, benzoate and biphenyl. It was found to inhibit heavily the elimination of some di- and trichlorinated biphenyls. In contrast, its influence on the elimination of more chlorinated congeners was much less significant.The authors are with the Department of Biochemical Technology, Faculty of Chemical Technology, Slovak Technical University, 812 37 Bratislava, Slovakia.     

Relative importance of the trophic and direct pathways on PCB contamination in the rotifer species Brachionus calyciflorus (Pallas)

To determine the contribution of food ingestion (trophic pathway) to PCB contamination of zooplankton in the river Meuse (Belgium), we used ¹⁴C-labelled algae (Dictyosphaerium ehrenbergianum) to measure ingestion and assimilation rates in the rotifer species Brachionus calyciflorus. When the concentration of algae in the culture medium varied from 20 10³ to 200 10³ algal cells ml^–1 (0.12 to 1.18 mg Cl^–1), the Brachionus calyciflorus ingestion rate varied from 0.25 ± 0.12 to 1.52 ± 0.43 ng C ind^–1 h^–1 at 15 °C and from 0.74 ± 0.17 to 5.93 ± 0.61 ng C ind^–1 h^–1 at 20 °C. The assimilation efficiency (ratio of the assimilation rate to the ingestion rate) measured in a culture medium containing 200 10³ algal cells ml^–1 was 55.7 ± 5.8%. Since the PCB concentration measured in the phytoplankton of the river Meuse is about 3 µg PCBs g^–1 D.W., the estimated PCB contamination of zooplankton ascribable to the trophic pathway ranges from 0.22 ± 0.17 to 1.31 ± 0.77 µg PCBs g^–1 D.W. at 15 °C and from 0.64 ± 0.34 to 5.10 ± 2.10 µg PCBs g^–1 D.W. at 20°C. The lower figure based on measurements effected at 20 °C is comparable to the actual level measured in zooplankton samples collected in the river Meuse (0.69 ± 0.20 µg PCBs g^–1 D.W.). The applicability of the formula used in our estimate was checked in a 48-hour in vitro experiment in which the rotifers were fed contaminated algae. The PCB accumulation measured in the rotifers was found to coincide with the calculated PCB contamination. Additional experiments were carried out to determine the contribution of the direct pathway to PCB contamination of zooplankton living in the river Meuse (0.02 µg PCBs l^–1 of water; average dissolved organic matter: 3 mg C 1^–1). The PCB concentration in zooplankton resulting from direct uptake of PCBs from the water was estimated at 0.19 ± 0.05 µg PCBs g^–1 D.W. These results show that in zooplankton living in polluted ecosystems, PCBs are likely to accumulate via the trophic pathway to concentrations up to 30 times higher than by direct contamination. Furthermore, our estimates of PCB contamination via the trophic pathway coincide quite well with actual concentrations measured in situ.     

The Effect of Sodium Azide on the Thermotolerance of the Yeasts Saccharomyces cerevisiae and Candida albicans

The addition of sodium azide (a mitochondrial inhibitor) at a concentration of 0.15 mM to glucose-grown Saccharomyces cerevisiae or Candida albicans cells before exposing them to heat shock increased cell survival. At higher concentrations of azide, its protective effect on glucose-grown cells decreased. Furthermore, azide, even at low concentrations, diminished the thermotolerance of galactose-grown yeast cells. It is suggested that azide exerts a protective effect on the thermotolerance of yeast cells when their energy requirements are met by the fermentation of glucose. However, when cells obtain energy through respiratory metabolism, the azide inhibition of mitochondria enhances the damage inflicted on the cells by heat shock.     

Biodegradation of polychlorinated biphenyls by plant cells

The PCB biodegradative ability of plant cells cultivated in vitro in media containing a mixture of PCB congeners, Delor 103, is demonstrated. For experiments we used submerged cultures of Armoracia rusticana, Solanum aviculare, Atropa bella-donna, transformed hairy root or embryogenic cultures of Solanum nigrum. Transformation of PCB was followed by gas chromatography after cultivations of the above-mentioned cultures with Delor 103 (10 mg 100 ml⁻¹). The overall PCB metabolizing capability and also degradation of individual congeners greatly differed from strain to strain. The highest capability to metabolize PCB was assayed with differentiated cultures of Solanum nigrum. Beside the capability of PCB degradation, total peroxidase activity in the medium and the cell extract was also followed. Differentiated or hairy root cultures exhibiting higher degradation abilities of PCB also showed increase of peroxidase activities.     

Lead phytoextraction from printed circuit computer boards by Lolium perenne L. and Medicago sativa L.

This work assessed the ability of Lolium perenne and Medicago sativa for extracting lead (Pb) from particulate printed circuit computer boards (PCB) mixed in sand with the following concentrations: 0.5, 1.0 and 1.5 g of PCB, and including a control treatment without PCB. The PCB were obtained from computers, and grinded in two particle sizes: 0.0594 mm (PCB1) and 0.0706 mm (PCB2). The PCB particle sizes at their corresponding concentrations were applied to L. perenne and M. sativa by using three experimental assays. In assay II, PCB2 affected the biomass production for both plants. For assay III, the PCB1 increased the biomass of M. sativa (236.5%) and L. perenne (142.2%) when applying either 0.5 or 1.0 g, respectively. In regards to phytoextraction, assay I showed the highest Pb-extraction by roots of L. perenne (4.7%) when exposed to 1.5 g of PCB1. At assay I, L. perenne showed a Pb-bioconcentration factor higher than 1.0 when growing at 0.5 g of PCB1, and when HNO₃ was used as digestion solution; moreover, in assay III both plants showed a Pb-translocation factor higher than 1.0. Therefore, Lolium perenne and Medicago sativa are able to recover Pb from electronic wastes (PCB).