Hydroxyl radical generation by redox cycling of some chemotherapeutic antibiotics

The anticancer drugs: adriamycin, farmorubicin and mitomycin C greatly enhance the generation of hydroxyl radicals (HO^.) from H₂O₂ in the presence of Co(II) ions (CoCl2) at pH 7.4 and 8, as measured by the deoxyribose assay. Catalase, hydroxyl radical scavengers (mannitol, cysteine, glutathione, thiourea, lactic dehydrogenase) inhibited the degradation of deoxyribose confirming that HO‐radicals are responsible for the degradation of the carbohydrate.     

基于谷胱甘肽结合作用定量研究微囊藻毒素的解毒代谢机制

基于谷胱甘肽(GSH)解毒作用探讨了微囊藻毒素-RR(MCRR)在不同动物肝脏和肾脏合作下的代谢机制。通过人工合成MCRR的谷胱甘肽代谢物(MCRR-GSH),腹腔注射至鲫鱼和大鼠体内,利用液相色谱串联质谱技术(LC-MS/MS)定量检测MCRR-GSH及其下游半胱氨酸代谢物(MCRR-Cys)在组织内的代谢动力学变化。在72 h的暴露实验中,实验组鲫鱼和大鼠体内均定量检测到MCRR-GSH和MCRR-Cys。MCRR-GSH在肾脏中的浓度显著高于其他组织(P0.05),鲫鱼和大鼠体内累积浓度分别是(0.161±0.001)和(0.116±0.005)μg·g~(-1)DW。同样的,MCRR-Cys主要分布于鲫鱼和大鼠的肾脏组织。鲫鱼肾脏中MCRR-Cys的浓度出现明显的波动,而肝脏和胆汁内的MCRR-Cys浓度却呈现出上升的趋势;大鼠肾脏内MCRR-Cys的浓度呈缓慢下降的趋势,浓度范围为(8.899±0.817)μg·g~(-1)DW至(3.336±0.263)μg·g~(-1)DW。基于以上结果推测,微囊藻毒素在肝脏和肾脏合作下的解毒过程为:MC在肝脏内经GSH结合作用生成的代谢物MC-GSH随血液循环转运至肾脏,在肾脏内MCGSH快速地转化为下游代谢物MC-Cys以促进排泄。     

Polarographic investigations on the complexation of cadmium and zinc by thiol peptides

Complex formation of Cd²⁺ and Zn²⁺ with thiol derivatives has been investigated by differential pulse polarography. The binding of Cd²⁺ and Zn²⁺ with cysteine (CySH), glutathione (GSH) and the model peptide N‐acetyl‐cysteine‐methylamide (ASH) reveals different stoichiometry. Thus, Cd²⁺ forms 1:1 and 1:2 complexes with CySH while 1:2 and 1:4 complexes have been observed with GSH and ASH, respectively. Overall formation constants of Cd²⁺ with CySH (Iogβ ₂ 15.3) and with GSH (Iogβ5₂ 14.4) have been estimated using competitive complexation with nitrilotriacetic acid (NTA). Investigation of competition between Zn²⁺ and Cd²⁺ for the thiol complexation has underlined the role played by the amino group in CySH for the stabilization of Zn complexes in contrary to Cd complexes.     

Glutathione and associated enzymes in toxic cataractogenesis-selenite model

Glutathione, gamma-glutamyl cysteine synthetase (gamma -GCS) and glutathione reductase (GSH-R) activity were determined biochemically in the lens during various stages after subcutaneous administration of sodium selenite in multiple low dosages and single high dosages. The GSH concentration and gamma-GCS and GSH-R activity declined progressively after the selenite administration. The changes observed were discussed in relation to the possible role of selenite interaction with GSH and the enzymes.     

Metabolites of sulfur‐containing derivatives of the fungicides pentachloronitrobenzene and hexachlorobenzene in mammals,separated by capillary gas chromatography and identified by negative and positive Cl mass spectrometry

Biotransformation products of sulfur‐containing derivatives of pentachloronitro‐benzene and hexachlorobenzene in mammals, such as thiophenols, thioanisoles, phenols, anisoles and chlorinated benzenes have been identified by mass spectrometry of negative and positive ions produced by chemical ionisation. The capillary gas chromatograms of extracts from excreta of rats after application of N‐acetyl‐S‐(pentachlorophenyI)‐D,L‐cysteine, as well as of rabbits after application of S,S'‐(tetrachloro‐p‐phenylene)‐L,L'‐dicysteine, are shown.     

Glutathione and cysteine biosynthesis in two varieties of Abelmoschus esculentus in response to mine spoil

The extent of accumulation of some heavy metals and glutathione and cysteine levels in the roots and aerial plant parts in two genotypically different varieties of A. esculentus (KS404 and BO2) exposed to mine spoil were investigated. Glutathione (GSH) level in both the varieties on control sites increased from basal level to 155.15 nmol g(-1) dry weight (d.wt.), almost 1.5 fold on 30 day and attained a plateau within 60 day Mine spoil exposure of both the varieties decreased glutathione 1.13 fold (89.2 nmol g(-1) dry weight) during 60 day from its basal level. GSH concentration in shoots of these varieties increased accompanying growth contrary to roots where it finally declined 2 fold. Cysteine content in control plants increased 2 fold (31.6 nmol g(-1) dry weight) on 30 day and finally declined 1.38 fold (22.35 nmol g(-1) dry weight, at 60 day). Both the varieties, when exposed to mine spoil, showed enhanced cysteine content almost 2 fold during 30 day (50.95 nmol g(-1) dry weight) but failed to increase further Forshoots in both the varieties challenged with mine spoil, cysteine maxima reached late (15.2 nmol g(-1) dry weight, at 40 day) relative to control but the levels declined subsequently (11.85 nmol g(-l) dry weight). Contrary to GSH, cysteine content in roots of both the varieties responded positively to mine spoil as apparent from the 2.23 fold increase during 30 d than basal level although it lowered to a level of 12.85 nmol g(-1) dry weight finally at 60 day. Both the varieties accumulated almost maximum level of selected cations (Fe > Mn> Zn> Cu > Ni) during 30 day, but BO2 variety was significantly superior in this regard. Invariably high accumulation of such cations in roots over shoots indicated accumulation, retention or restricted translocation from root to shoot. The metal share of the edible part was just 6% of the plant load. Thus, present work reflects a genotypic differences in metal accumulation and that affected the major non-enzymatic traits or synthesis of sulthydryl compounds as well. The present results also indicate that metal tolerance is in part associated with anti-oxidant system activity.     

Free ‐ living animals as indicators of environmental pollution by chlorinated hydrocarbons

As some species of game and free‐living animals promptly respond to ecosystem damage by a reaction that is easy to interpret, they are important ecological indicators in assessing the total contamination of the environment. Consumers of the highest order like predators, predatory and fish‐eating birds are among the most important bioindicators of environmental contamination by chlorinated hydrocarbons and PCB.

Data on chlorinated hydrocarbons and PCB levels were collected throughout 1988–1992 and obtained from 16 species of free‐living animals from 9 protected areas of Slovakia. Muscle tissue, liver, kidney and egg samples were examined for HCB, HCH isomers, DDT and its analogues, and PCB.

Levels of organic chlorinated contaminants reveal a decreasing tendency and thus cease to be a toxicologically important risk factor in game, with the exception of predatory birds, as significant values have been recorded in their eggs and reproductive organs.     

Polycyclic aromatic hydrocarbons in surface sediments held in experimental mesocosms

Sediment was collected from three locations along a pollution gradient in Narragansett Bay and transplanted to controlled mesocosms. Total hydrocarbons and eleven individual polycyclic aromatic hydrocarbons (PAHs) were measured in these sediments over a period of 394 days. Total hydrocarbon concentrations increased in the “relatively uncontaminated”; (Rhode Island Sound) sediment that was held in the mesocosms, but did not change in the two other sediments. The concentrations of four PAHs: naphthalene, 2‐methyl naphthalene, 1‐methyl naphthalene and biphenyl, decreased in the “contaminated”; (Providence River) sediment during the experiment and the calculated half‐lives for these compounds were 287, 353, 321 and 333 days, respectively.     

Dissolved and dispersed petroleum hydrocarbons in the Aegean Sea

The distribution and transportation of Dissolved and Dispersed Petroleum Hydrocarbon (DDPH) were investigated in the Aegean Sea with the hydrodynamics of the water masses of the region. It is clear that distribution of this pollutant is strongly affected by physical dynamics of environment. The data were collected during cruises in November 1994, in the framework of National Marine Measurement and Monitoring Programme in the Aegean Sea. In the present study additionally the Chlorophyll‐a was measured fluorometrically and there is good correlation between petroleum hydrocarbon and chlorophyll‐a in the Aegean Sea. DDPH data was used to search origin of hydrocarbons: biogenic or non biogenic.     

Vertical infiltration of fuel oil hydrocarbons in an agricultural soil

The influence of rainfall, air temperature and soil moisture on the vertical mobility in the soil of fuel oil hydrocarbons (HC) was investigated in a field experiment. A controlled spreading of fuel oil (nC10‐nC25) was performed at a rate of 5 L HCm^‐2 on an agricultural soil in summer and in winter. Concentration, chemical composition of HC and soil moisture were regularly determined at different soil depths between 0 and 140 cm, 1 h, 3, 8and 15 days (d) after the spreading of oil. Sorption of hydrocarbons onto the organo‐mineral matrix of the soil was studied in laboratory experiments. The results showed that in summer, with an air temperature of 24°C and without water leaching in the soil profile, 65% of the initial HC remained trapped in the 0–140 cm soil layer, about 20% of the HC volatilized and around 15% migrated deeper. A vertical selective migration of the lightest (nC10‐nC15) HC (naphthas) was shown lSd after the spreading of fuel oil. Naphthas progressively reached the 120–140 cm soil layers whereas the heavy fractions of oil (nC17‐nC25) migrated and concentrated in the 0–60 cm soil layers. In winter, when soil was regularly watered by rainfalls and at low air temperatures, only 47% of the initial HC remained in the 0–140 cm profile after 15 d. A fast vertical infiltration of naphthas occurred within the first 3 d. After 15 d, all HC were detected in the same relative amounts as in the initial oil in the whole profile. Volatilization was negligible in winter and an increase in the migration of total oil at depth in the soil profile was shown. As inferred from the laboratory experiments, the high soil moisture led to the decrease in HC sorption on the organo‐mineral matter of the soil.     

HPLC analysis of nonprotein thiols in planktonic diatoms: pool size,redox state and response to copper and cadmium exposure

A sensitive method was developed to analyze low molecular weight thiols involved in metal homeostasis and detoxification in phytoplankton. The aims of this study were to (1) separate and measure all relevant thiols in a single HPLC run, (2) measure redox states of the thiols and (3) identify specific responses of thiols (pools, redox) to heavy metals by testing diatoms with different metal tolerances (Ditylum brightwellii, Phaeodactylum tricornutum, Skeletonema costatum andThalassiosira pseudonana). Copper or cadmium were dosed at a maximum tolerable, species-dependent level, to exponential phase cells growing in artificial medium (14 salinity). Loss of cell viability was monitored by the decrease of fluorescein fluorescence after a 24-h metal exposure. Thiols in extracts of exposed cells and controls were labeled with monobromobimane. Picomoles of cysteine, glutathione, -glutamylcysteine and phytochelatins (PCs) were detected and separated by reversed-phase high performance liquid chromatography. Cysteine increased in all species after metal exposure. The species with the largest glutathione pools in the control (P. tricornutum) synthesized the largest PC pools upon metal exposure, however, at a 40% loss of glutathione. A considerable increase of glutathione was observed inD. brightwellii upon metal exposure. However, it produced little PC (and only with Cu). In controls ( 3 pM Cu²⁺), PC₂ was detectable inS. costatum, P. tricornutum andT. pseudonana. Oxidized thiol fractions were recovered by the reductants DTT and TCEP, both performing identically. Compared to the other thiols, cysteine had low redox ratios. InD. brightwellii glutathione and PC redox ratios were lower than inP. tricornutum, S. costatum andT. pseudonana. It was expected that Cu-induced oxidative stress would decrease the thiol redox ratios, however, this was not observed.This is Communication No. 2172 of NIOO.     

Mechanism of toxicity of zinc to the marine diatomNitzschia closterium

The effect of zinc on cell division, photosynthesis, ultrastructure, respiration, ATP levels, mitochondrial electron-transport chain (ETC)-activity, total thiols and glutathione in the marine diatomNitzschia closterium (Ehrenberg) W. Smith was investigated. Although 65µg Zn 1^–1 halved the cell division rate, photosynthesis and respiration were unaffected by zinc concentrations up to 500µg Zn 1^–1. Most of the zinc associated with the cells was bound at the cell surface, with only 3 to 4% of this extracellular zinc penetrating the cell membrane. Once inside the cell, zinc exerted its toxicity at a number of sites. Increased ATP production and ETC activity were observed in zinc-treated cells. Zinc also enhanced cellular thiols (SH) and total glutathione, and zinc toxicity was reversible by the addition of thiol compounds such as cysteine. Zinc-thiol binding may be a detoxification mechanism for the cell. It is suggested that increased ATP production may provide the energy required for increased glutathione synthesis at the expense of other energy-requiring processes including cell division. The mechanisms of toxicity of ionic zinc and copper toN. closterium were compared.     

In vitro Methylation of Arsenite in Flemish giant rabbit Cytosol

The formation of ⁷⁴As‐monomethyIarsonic acid and ⁷⁴As‐dimethylarsinic acid from carrier‐free radiolabelled ⁷⁴As‐arsenite was evaluated in an assay mixture containing 17.6% liver cytosol from the Flemish Giant rabbit. The optimal incubation temperature and pH were respectively, 39°C and 7.6. After a 2h incubation about 90% of ⁷⁴As was protein bound. Protein bound ⁷⁴As was released by hot 2 M HNO₃ (1 min, 110°C). The treatment did not destroy methylated As‐species. Up to 70% of the carrier‐free ⁷⁴As‐arsenite was methylated. Liver cytosol was stored, without loss of activity, in liquid nitrogen in the presence of 2 mM glutathione. The optimal s‐adenosyl‐methionine concentration was 1.7mM. Formation of ⁷⁴As‐monomethylarsonic acid and ⁷⁴As‐dimethylarsinic acid increased up to a glutathione concentration of respectively 10 and 20 mM. Methylation also went through in the presence of other reducing agents and proved to be ATP dependent.     

Microbiological degradation of polycyclic aromatic hydrocarbons: Anthracene,benzo(K)fluoranthene,dibenzothiophene, benzo(H) quinoline and 2‐nitronaphthalene

Biodegradation experiments of various polycyclic aromatic hydrocarbons were studied with mixed bacteria culture under aerobic conditions. An easy‐to‐handle clean‐up procedure was developed for PAH and their metabolites simultaneously as well as a gc‐ms‐method to identify and quantify these compounds.

Anthracene and dibenzothiophene are completely degradable in an aqeous system, whereas biodegradation of benzo(k)fluoranthene and benzo(h)quinoline is possible only in an oil‐in‐water‐system with dodecane as cosubstrate. No degradation of nitronaphthalene was observed in aqueous systems. New metabolites are 2,3‐dihydroxybenzothiophene, hydroxybenzothiophenecarbonic acid and benzothiophenequinone for dibenzothiophene and hydroxyfluoranthenic acid for benzo(k)flouranthene. Whereas the former metabolites are degradable under the experimental conditions, the latter accumulates during the degradation experiment.

The results are important for microbiological wastewater treatment, since knowledge of biodegradation processes is indespensable for the successful treatment of PAH‐containing wastewater.     

Fate of petroleum hydrocarbons taken up from food and water by the blue crab Callinectes sapidus

Radiolabeled paraffinic and aromatic hydrocarbons, including benzopyrene, fluorene, naphthalene, methylnaphthalene, methylcholanthrene, hexadecane, heptadecane and dotriacontane, were taken up from food and water by the blue crab Callinectes sapidus. In 2 days, approximately 10% of the benzopyrene and fluorene were taken up from the water when their concentrations were 2.5 and 30 g/l, respectively. When given food with radiolabeled hydrocarbons, 2 to 10% of the hydrocarbons were assimilated by the carbs, with the remainder excreted. After uptake of hydrocarbon from water or food, a major pathway for the elimination of hydrocarbon and metabolites was through fecal material. All hydrocarbons used in the study were metabolized, with similar rates for both paraffinic and aromatic hydrocarbons. More than 50% of the radioactivity assimilated by the crabs was in the hepatopancreas, suggesting that the hepatopancreas was the site of hydrocarbon metabolism. Twenty-five days aftex exposure to radiolabeled hydrocarbons, radioactivity was found only in the hepatopancreas. The hepatopancreas contained highly polar hydrocarbon metabolites, including dihydroxy-compounds and their conjugates, while blood contained both monohydroxy-and dihydroxy-compounds. No evidence was found of storage of hydrocarbons by any of the crab tissues.     

Anaerobic digestion of sludge by different pretreatments: Changes of amino acids and microbial community

•Tryptophan protein, and aromatic protein I/II were the key identified proteins. •Cysteine was more correlated with methane production than other amino acids. •The presence of cysteine can promote methane production and degradation of VFAs. •The presence of cysteine can lower ORP and increase biomass activity. •Predominant Tissierella and Proteiniphilum were noted in pretreated sludge samples. Many studies have investigated the effects of different pretreatments on the performance of anaerobic digestion of sludge. However, the detailed changes of dissolved organic nitrogen, particularly the release behavior of proteins and the byproducts of protein hydrolysis-amino acids, are rarely known during anaerobic digestion of sludge by different pretreatments. Here we quantified the changes of three types of proteins and 17 types of amino acids in sludge samples solubilized by ultrasonic, thermal, and acid/alkaline pretreatments and their transformation during anaerobic digestion of sludge. Tryptophan protein, aromatic protein I, aromatic protein II, and cysteine were identified as the key dissolved organic nitrogen responsible for methane production during anaerobic digestion of sludge, regardless of the different pretreatment methods. Different from the depletion of other amino acids, cysteine was resistant to degradation after an incubation period of 30 days in all sludge samples. Meanwhile, the “cysteine and methionine metabolism (K00270)” was absent in all sludge samples by identifying 6755 Kyoto Encyclopedia of Genes and Genomes assignments of genes hits. Cysteine contributed to the generation of methane and the degradation of acetic, propionic, and n-butyric acids through decreasing oxidation-reduction potential and enhancing biomass activity. This study provided an alternative strategy to enhance anaerobic digestion of sludge through in situ production of cysteine.     

Isolation of petroleum and chlorinated hydrocarbons from the same extract

Sequential analysis is an extremely powerful technique for complex samples analysis. For determination of petroleum and chlorinated hydrocarbons, the same sample is divided into two parts. One part is utilized to extract the petroleum hydrocarbons and the second part is used to extract the organochlorine compounds because petroleum hydrocarbons usually appear in higher concentrations and interfering with organochlorine compounds. This analysis method required more solvent, time, and more sample volume. The analytical strategy, in this paper, involves the use of effective and efficient but simple techniques for petroleum hydrocarbons and chlorinated hydrocarbons separation. A clean‐up procedure is presented, by which these compounds are isolated from aliphatic as well as polyaromatic hydrocarbons through a combination of HPLC and adsorption chromatography on a florisil microcolumn. Mass spectrometric identification was used as confirmation method. GLC equipped with ECD and FID for organochlorine and petroleum compounds analysis, respectively.     

The role of sulphur in cadmium(II) ions detoxification demonstrated in in vitro model: Dionaea muscipula Ell.

Fertilization by phosphorus and also nitrogen fertilizers are the main source of cadmium soil contamination. Cadmium is relatively easily taken up by plants and then consequently enters their food chain. We investigated whether the cultivation medium with or without sulphates supplement affects the synthesis of protective low molecular mass thiols such as cysteine, reduced glutathione, oxidized glutathione and phytochelatins (PC2) in Dionaea muscipula treated with cadmium(II) ions at 0, 1, 5, 10, 25, 50, 100, 250, 500 and 1,000 μM for 6 weeks. The plants cultivated in the presence of sulphates showed higher cadmium tolerance due to faster growth and lesser number of necrosis hallmarks. Further, we utilized liquid chromatography with electrochemical detection for determination of the thiols in plant tissues. In the case of the plants cultivated on media supplemented with sulphates for 6 weeks the average PC2 level was 2,830 ng/g of fresh weight (FW). However, the plants cultivated on media without the presence of sulphates on average contained 1,160 ng PC2/g FW. Results obtained showed the positive effect of sulphur supplementation in cadmium detoxification processes in plants. In addition to thiol content, we also determined level of majority secondary metabolite of Venus flytrap, naphthoquinone plumbagin. Generally, the presence of sulphates in the media enhanced the protective mechanism and did not affect directly the synthesis of secondary metabolite plumbagin.     

Fluorescence studies of dye displacement from DNA by chromium(III) complexes: Evidence for cation induced DNA condensations

The interactions of 10 different chromium(III) complexes with isolated calf thymus DNA have been analysed by studying the electronic and fluoresence spectra of intercalated ethidiumbromide. Triply charged cationic complexes including: [Cr(urea)₆]Cl₃.3H₂O, [Cr(1,10‐phenanthroline)₃](ClO₄)₃.2H₂O, [Cr(2,2'‐bipyridyl)₃] (ClO₄)₃.2H₂O, [Cr(ethylendiamine)₃]Cl₃.3.5H₂O and [Cr(NH₃)₆](NO₃)₃ displaced the dye from DNA. Similar effects were observed in experiments using the non‐intercalating dye bisbenzimidazole ("Hoechst 33258"). However, singly charged cationic, anionic and uncharged chromium(III) complexes such as: cis‐[Cr(1,10‐phenanthroline)₂Cl₂]Cl.2H₂O, cis‐[Cr(2,2'‐bipyridyl)₂Cl₂]Cl.2H₂O, [Cr(glutathione)₂]Na₂, [Cr(cysteine)₂]Na.2H₂O and [Cr(glycine)₃] were unable to displace both ethidiumbromide and bisbenzimidazole from DNA. There was no evidence for the formation of co‐ordinate bonds between chromium(III) and DNA for any of the above complexes. The charge and type of ligand are important in controlling the interaction of chromium(III) with isolated DNA in vitro. Our findings indicate that the outer sphere interaction of a chromium(III) complex with DNA is weak and unlikely to be the mechanism by which chromate causes DNA impairments in vivo and in vitro.     

Cellular response of mouse splenocytes to heavy metals exposure

Among the environmental contaminants recognised for their toxicity and their global presence, heavy metals are certainly a major concern. They can elicit a number of immunomodulatory effects leading ultimately to an enhanced susceptibility (sensitivity) of immune cells to microbial agents and the appearance of neoplastic diseases and autoimmune phenomena. Heavy metals also provoke changes in the function(s) of immune cells. A striking biological effect of heavy metals is the induction of intracellular thiols (cysteine, glutathione, metallothioneins). Thiols are involved in many physiological processes, including protection from free radical damage and detoxification of chemicals. The purpose of this study was to assess the differences of susceptibility (sensitivity) in both pre-activated (concanavalin A was used) and non-pre-activated cells in the presence of heavy metals. Five were evaluated on murine splenocytes. The lymphoblastic proliferation test was performed for lymphocytes and a phagocytosis test for macrophages. Data showed that the levels of thiols in the pre-activated cells are greater than non-pre-activated cells following exposure to various heavy metals; macrophages were more resistant than lymphocytes to the toxic effects of heavy metals, and pre-activated cells were more resistant than cells at rest. One possible explanation is that macrophages produce more thiols than lymphocytes and this provides an increased protection from the deleterious effects of heavy metals.