Biochemical assay by immobilized enzymes and a mass spectrometer.

By counting the volatile molecules produced by an immobilized-enzyme catalyzed reaction which is interfaced to a mass spectrometer via a semi-permeable membrane, a general approach to biochemical measurement and detection is obtained which offers the potential of high sensitivity, specificity and speed. In combination with molecule microscopy, this method should allow, for example, a mapping of suitable enzyme distributions in non-stained and non-fixed tissue slices. Immobilized urease (urea amidohyrdrolase, EC 3.5.1.5) was used to assay urea using CO2 as the volatile product, and alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) was used to assay NADH using ethanol as the volatile product.     

Prevalidation study of the BALB/c 3T3 cell transformation assay for assessment of carcinogenic potential of chemicals

The cell transformation assays (CTAs) have attracted attention within the field of alternative methods due to their potential to reduce the number of animal experiments in the field of carcinogenicity. The CTA using BALB/c 3T3 cells has proved to be able to respond to chemical carcinogens by inducing morphologically transformed foci. Although a considerable amount of data on the performance of the assay has been collected, a formal evaluation focusing particularly on reproducibility, and a standardised protocol were considered important. Therefore the European Centre for the Validation of Alternative Methods (ECVAM) decided to coordinate a prevalidation study of the BALB/c 3T3 CTA. Three different laboratories from Japan and Europe participated. In the study the following modules were assessed stepwise: test definition (Module 1) consisted of the standardisation of the protocol, the selection of the cell lineage, and the preparation of a photo catalogue on the transformed foci. The within-laboratory reproducibility (Module 2) and the transferability (Module 3) were assessed using non-coded and coded 3-methylcholanthrene. Then, five coded chemicals were tested for the assessment of between-laboratory reproducibility (Module 4). All three laboratories obtained positive results with benzo[a]pyrene, phenanthrene and o-toluidine HCl. 2-Acetylaminofluorene was positive in two laboratories and equivocal in one laboratory. Anthracene was negative in all three laboratories. The chemicals except phenanthrene, which is classified by IARC (http://monographs.iarc.fr) as group 3 "not classifiable as to its carcinogenicity to human", were correctly predicted as carcinogens. Further studies on phenanthrene will clarify this discrepancy. Thus, although only a few chemicals were tested, it can be seen that the predictive capacity of the BALB/c 3T3 CTA is satisfactory. On the basis of the outcome of this study, an improved protocol, incorporating some changes related to data interpretation, has been developed. It is recommended that this protocol be used in the future to provide more data that may confirm the robustness of this protocol and the performance of the assay itself. During the study it became clear that selecting the most appropriate concentrations for the transformation assay is crucial.     

The assay of in-situ activities of sulphate-reducing bacteria in a laboratory marine corrosion model

A critical reappraisal of the [³⁵S] sulphate reduction assay as applied to studies of microbial corrosion has identified sulphate limitation and the production of both acid volatile and nonacid volatile sulphides as key factors which must be incorporated into the assay and into the interpretation of its findings. Application of the modified assay to a laboratory corrosion model gave data reinforcing the fundamental role of environmental factors, notably oxygen, in determining the nature and extent of the corrosion process.     

Metabolism of phenanthrene by house fly CYP6D1 and dog liver cytochrome P450

Polycyclic aromatic hydrocarbons (PAHs) are a ubiquitous class of environmental contaminants. The compound phenanthrene is a model PAH. A novel fluorometric method for measuring phenanthrene metabolism in vitro was developed and verified with direct measurement of [14C]phenanthrene using dog liver microsomes. The fluorometric assay and direct measurement of [14C]phenanthrene metabolism were used to show that CYP6D1, a house fly cytochrome P450, is the major house fly P450 involved in phenanthrene metabolism. Phenanthrene was metabolized by microsomes from the LPR strain of house fly that overexpresses CYP6D1, but metabolism was not observed in the CS strain that has a lower level of CYP6D1. Furthermore, the majority of phenanthrene metabolism was inhibited by a CYP6D1-specific antibody. This study increases the number of known substrates of CYP6D1 and identifies polyaromatic hydrocarbons as potential substrates of CYP6D1. The utility of CYP6D1 as an agent in bioremediation and the utility of the new fluorometric assay for understanding PAH metabolism in insects and mammals are discussed.     

Evaluation of the genotoxic potential of some microbial volatile organic compounds (MVOC) with the comet assay,the micronucleus assay and the HPRT gene mutation assay

Microbial volatile organic compounds (MVOC), metabolites of fungi detected in indoor moulds and in working places in compost facilities are considered as a potential health hazard. Their toxicological relevance, however, is largely unknown and data are rare. The aim of this study was to evaluate in vitro the genotoxic, clastogenic and mutagenic potential of same typical MVOC. For the study of DNA damage human lung carcinoma epithelial A549 cells, V79 Chinese hamster fibroblasts and human peripheral blood cells were exposed and subjected to the alkaline comet assay (single cell gel test). Taking the Chinese hamster V79 cell line as a target clastogenic effects were studied by the micronucleus test and mutagenic effects by the hypoxanthine-guanine-phosphoribosyl transferase gene mutation test (HPRT test). The cytogenic effects of MVOC were assessed by a clonogenic assay using the A549 cell line. The alkylating agent methyl methanesulfonate (MMS) was taken as a positive control. The results indicate that MVOC induced DNA damage is only seen in conditions in which also cytotoxic effects are observed. Clastogenic and mutagenic effects could not be detected.     

Evaluation of the genotoxic potential of some microbial volatile organic compounds (MVOC) with the comet assay, the micronucleus assay and the HPRT gene mutation assay.

Microbial volatile organic compounds (MVOC), metabolites of fungi detected in indoor moulds and in working places in compost facilities are considered as a potential health hazard. Their toxicological relevance, however, is largely unknown and data are rare. The aim of this study was to evaluate in vitro the genotoxic, clastogenic and mutagenic potential of same typical MVOC. For the study of DNA damage human lung carcinoma epithelial A549 cells, V79 Chinese hamster fibroblasts and human peripheral blood cells were exposed and subjected to the alkaline comet assay (single cell gel test). Taking the Chinese hamster V79 cell line as a target clastogenic effects were studied by the micronucleus test and mutagenic effects by the hypoxanthine-guanine-phosphoribosyl transferase gene mutation test (HPRT test). The cytogenic effects of MVOC were assessed by a clonogenic assay using the A549 cell line. The alkylating agent methyl methanesulfonate (MMS) was taken as a positive control. The results indicate that MVOC induced DNA damage is only seen in conditions in which also cytotoxic effects are observed. Clastogenic and mutagenic effects could not be detected.     

Degradation of phenanthrene by the endophytic fungus <Emphasis Type="Italic">Ceratobasidum stevensii</Emphasis> found in <Emphasis Type="Italic">Bischofia polycarpa</Emphasis>

Some strains of white rot fungi, non-lignolytic fungi and litter-decomposing basidiomycetes have been recognized as PAH degraders. The purpose of our research was to enlarge the scope of PAH-degrading fungi and explore the huge endophytic microorganism resource for bioremediation of PAHs. In this study, phenanthrene was used as a model PAHs compound. Nine strains of endophytic fungi isolated from four kinds of plant from Eupharbiaceae were screened for degradation of phenanthrene. The endophytic fungus Ceratobasidum stevensii (strain B6) isolated from Bischofia polycarpam showed high degradation efficiency and was selected for further studies. Into the fungal culture, 100 mg l⁻¹ phenanthrene was added, and after 10 days of incubation, about 89.51% of the phenanthrene was removed by strain B6. Extracellular ligninolytic enzyme activities of strain B6 were tested. The results showed that manganese peroxidase [MnP] was the predominant ligninolytic enzyme and that its production was greatly induced by the presence of phenanthrene. To confirm the involvement of MnP in phenanthrene degradation, promotion and inhibition studies on MnP in different concentration level of Mn²⁺ and NaN₃ were performed. Additionally, fungal mycelium-free and resuspended experiments were carried out. The results showed no apparent correlation between MnP activity and phenanthrene degradation. The mycelium and fresh medium were the crucial factors affecting the degradation of phenanthrene. To date, this is the first report on PAH degradation by Ceratobasidum stevensii. This study suggests that endophytic fungi might be a novel and important resource for microorganisms that have PAH-degrading capabilities.     

A simple pentose assay for biomass conversion studies

Summary A colorimetric method was modified for monitoring pentose release and utilization in the hydrolysis and fermentation of biomass substrates to fuels and chemicals. The proposed assay was specific for pentose monomers. Quantitation of pentoses by the assay method was not significantly interfered by other lignocellulosic components, common fermentation medium ingredients, and major volatile fermentation products encountered in biomass conversion processes. The assay procedure did not require sample pretreatment (e.g. deproteinization, desalting, or furfural extraction). Sugar estimation basing on the present assay correlated well with conventional sugar analysis by high performance liquid chromatography.     

Mutagenicity of benzyl chloride in the Salmonella/microsome mutagenesis assay depends on exposure conditions

Benzyl chloride (BCl) is a clear yellowish, volatile liquid that is widely used as an intermediate for the production of benzyl alcohol and benzyl compounds used in perfumery, dyes and pharmaceuticals. In previous studies BCl has shown weak and inconsistent mutagenicity in the Salmonella/microsome mutagenesis assay (Ames test). The aim of the present study was to investigate the potential mutagenic activity of BCl using modifications of the standard Ames test in order to adapt the method to the volatile nature of the test compound. Tests were performed using (a) the standard plate-incorporation method, (b) incubation of the treated plates in closed containers, (c) a vaporization-diffusion method to expose Ames test plates to volatilised BCl and (d) the pre-incubation method. Using the standard plate-incorporation method, BCl showed no (in the absence of metabolic activation) or very weak (in the presence of metabolic activation) mutagenic activity in Salmonella typhimurium tester strain TA 100. The use of the pre-incubation method did not improve detection of mutagenic activity of BCl. The use of closed containers significantly increased the response, but the most marked response was obtained by testing BCl in volatilised form in the vaporization-diffusion method. Using the latter approach there appeared to be less toxicity of the BCl treatments to the tester bacteria. Our findings suggest that BCl may show greater mutagenic activity in the gaseous phase. This work underlines the importance of using appropriate methods for the evaluation of volatile compounds. The modifications described here are easy to realize in practice and should prove useful for the investigation of other volatile materials or atmospheric contaminants.     

Genotoxic effects of volatile organic compounds in a chemical factory as evaluated by the Tradescantia micronucleus assay and by chemical analysis

The clastogenic effects of volatile organic compounds in the workplace air of a chemical factory were studied by means of the Tradescantia micronucleus (Trad-MCN) assay and chemical analysis. Sampling was performed at a chemical factory producing PVC film in Cheong-ju, South Korea. Inflorescences of Tradescantia BNL 4430 were placed for 2, 6, and 9 h at the height of 1.40 m at two locations in the workplace and one outdoor of the chemical industry. Air samplings were conducted in the same places and the collected tube samples were analyzed by automatic thermal desorption/gas chromatography/mass spectrometry (ATD/GC/MS). The frequencies of micronuclei in specimens exposed for 2 h in sites 1-3 were 6.13 +/- 0.47, 5.40 +/- 1.60, and 2.93 +/- 0.43 MCN per 100 tetrads, respectively. GC/MS analysis proved the presence of various volatile organic compounds such as trichloroethylene, toluene, ethyl benzene, (m, p, o)-xylene, styrene, 1,3,5-trimethyl benzene, and 1,2,4-trimethyl benzene. Mean values of toluene measured by 2 h sampling in sites 1-3 were 1946.6, 1368.3, and 340.1 microg/m3, respectively. The toluene concentrations in sites 1 and 2 were at least four to six times higher than that in site 3. The micronucleus frequencies increased with exposure time. In addition, there was a correlation between the micronucleus frequencies and toluene concentration in the air (R2 = 0.96). The results of this in situ monitoring proved the applicability of the Trad-MCN assay combined with chemical analysis for monitoring genotoxic chemicals in the work environment.     

Evaluation of the antioxidant activity of flavonoids by "ferric reducing antioxidant power" assay and cyclic voltammetry

Flavonoids, naturally occurring phenolic compounds, have recently been studied extensively for their antioxidant properties. The structure-antioxidant activity relationships (SAR) of flavonoids have been evaluated against different free radicals, but "ferric reducing antioxidant power" (FRAP) assay, which determines directly the reducing capacity of a compound, has not been used for this purpose. In this study, the antioxidant activities of 18 structurally different flavonoids were evaluated by FRAP assay modified to be used in 96-well microplates. Furthermore, their oxidation potentials were also measured, which were in the range of +0.3 V (myricetin) to +1.2 V (5-hydroxy flavone) and were in good agreement with FRAP assay results. Quercetin, fisetin and myricetin had the lowest oxidation potentials and appeared the most active compounds in FRAP assay and were 3.02, 2.52 and 2.28 times more active than Trolox, respectively. Indications were found that the o-dihydroxy structure in the B ring and the 3-hydroxy group and 2,3-double bond in the C ring give the highest contribution to the antioxidant activity.     

Genotoxicity of the volatile anaesthetic desflurane in human lymphocytes in vitro, established by comet assay

The aim of the present study was to estimate the genotoxicity of desflurane, applied as a volatile anaesthetic. The potential genotoxicity was determined by the comet assay as the extent of DNA fragmentation in human peripheral blood lymphocytes in vitro. The comet assay detects DNA strand breaks induced directly by genotoxic agents as well as DNA fragmentation due to cell death. Another anaesthetic, halothane, already proved to be a genotoxic agent, was used as a positive control. Both analysed drugs were capable of increasing DNA migration in a dose-dependent manner under experimental conditions applied. The results of the study demonstrated that the genotoxicity of desflurane was comparable with that of halothane. However, considering the pharmacodynamics of both drugs, the genotoxic activity of desflurane may be connected with a less harmful effect on the exposed patients or medical staff.     

Insights into the genome and proteome of <Emphasis Type="Italic">Sphingomonas paucimobilis</Emphasis> strain 20006FA involved in the regulation of polycyclic aromatic hydrocarbon degradation

In order to study the mechanisms regulating the phenanthrene degradation pathway and the intermediate-metabolite accumulation in strain S. paucimobilis 20006FA, we sequenced the genome and compared the genome-based predictions to experimental proteomic analyses. Physiological studies indicated that the degradation involved the salicylate and protocatechuate pathways, reaching 56.3% after 15 days. Furthermore, the strain degraded other polycyclic aromatic hydrocarbons (PAH) such as anthracene (13.1%), dibenzothiophene (76.3%), and fluoranthene. The intermediate metabolite 1-hydroxy-2-naphthoic acid (HNA) accumulated during phenanthrene catabolism and inhibited both bacterial growth and phenanthrene degradation, but exogenous-HNA addition did not affect further degradation. Genomic analysis predicted 126 putative genes encoding enzymes for all the steps of phenanthrene degradation, which loci could also participate in the metabolism of other PAH. Proteomic analysis identified enzymes involved in 19 of the 23 steps needed for the transformation of phenanthrene to trichloroacetic-acid intermediates that were upregulated in phenanthrene cultures relative to the levels in glucose cultures. Moreover, the protein-induction pattern was temporal, varying between 24 and 96 h during phenanthrene degradation, with most catabolic proteins being overexpressed at 96 h—e. g., the biphenyl dioxygenase and a multispecies (2Fe–2S)-binding protein. These results provided the first clues about regulation of expression of phenanthrene degradative enzymes in strain 20006FA and enabled an elucidation of the metabolic pathway utilized by the bacterium. To our knowledge the present work represents the first investigation of genomic, proteomic, and physiological studies of a PAH-degrading Sphingomonas strain.     

Determination of monod kinetic coefficients for volatile hydrophobic organic compounds

A new procedure is presented to determine Monod kinetic coefficients and the microbial yield coefficient for volatile hydrophobic compounds such as phenanthrene. Batch experiments were conducted with a mixed culture capable of degrading phenanthrene. The phenanthrene disappearance and carbon dioxide production were monitored with time. A maximum likelihood estimator was formulated to fit the set of equations that describe the system to the measured data. The model takes into account a number of processes such as partition onto the apparatus, volatilization, and partition onto the biomass. The parameters required to describe these processes were obtained by independent experiments. The yield coefficient could be determined within a small range. However, the specific growth rate and the half-saturation constant were found to vary widely, with pairs of them describing the system adequately. It was shown that partition and volatilization processes can significantly affect the determination of the yield and Monod kinetic coefficients and need to be taken into account. (c) 1996 John Wiley & Sons, Inc.     

The use of a hydroxylapatite-filter steroid receptor assay method in the study of the modulation of androgen receptor interaction

Receptors for androgen, estrogen, and glucocorticoid can be assayed by hydroxylapatite adsorption of the radioactive steroid-receptor complex and washing of the adducts on membrane filters mounted on a multiple filter holder. The method is economical, very rapid and sensitive. This new receptor assay method was used to study the modulation of androgen receptor of rat ventral prostate by metal ions, thiols, and ligand structure. The interaction of androgen with the naked receptor is inhibited by 10 microM ZnCl2, CdSO4, or CuSO4 but this inhibition is competed by androgen and is reversed by DTT. The androgen-receptor complex is less sensitive to divalent metal ions but Zn2+, at 3 mM, appears to alter the conformation of the receptor and promote the release of androgen. Certain phenanthrene derivatives exhibited striking structural specificities in their ability to compete with radioactive androgen for binding to the prostate receptor. The results suggest that the receptor has binding preference toward individual ring structure in the steroid.     

Phenanthrene degradation by bacteria of the genera <Emphasis Type="Italic">Pseudomonas</Emphasis> and <Emphasis Type="Italic">Burkholderia</Emphasis> in model soil systems

Degradation of phenanthrene by strains Pseudomona, Moscow, KMK, 2004simova, I.A. and Chernov, I.s putida BS3701 (pBS1141, pBS1142), Pseudomonas putida BS3745 (pBS216), and Burkholderia sp. BS3702 (pBS1143) were studied in model soil systems. The differences in accumulation and uptake rate of phenanthrene intermediates between the strains under study have been shown. Accumulation of 1-hydroxy-2-naphthoic acid in soil in the course of phenanthrene degradation by strain BS3702 (pBS1143) in a model system has been revealed. The efficiency of phenanthrene biodegradation was assessed using the mathematical model proposed previously for assessment of naphthalene degradation efficiency. The efficiency of degradation of both phenanthrene and the intermediate products of its degradation in phenanthrene-contaminated soil is expected to increase with the joint use of strains P. Putida BS3701 (pBS1141, pBS1142) and Burkholderia sp. BS3702 (pBS1143).     

Mutagenicity of phenanthrene and phenanthrene K-region derivatives.

Phenanthrene and 9 K-region derivatives, most of them potential metabolites of phenanthrene, were tested for mutagenicity by the reversion of histidine-dependent Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100 and the rec assay with Bacillus subtilis H17 and M45. The strongest mutagenic effects in the reversion assay were observed with phenanthrene 9,10-oxide, 9-hydroxyphenanthrene and N-benzyl-phenanthrene-9,10-imine. Interestingly, the mutagenic potency of the arene imine was similar to that of the corresponding arene oxide. This is the first report on the mutagenicity of arene imine. The mutagenic effects of all these phenanthrene derivatives were much weaker than that of the positive control benzo[a]pyrene 4,5-oxide. Even weaker mutagenicty was found with cis-9,10-dihydroxy-9,10-dihydrophenanthrene and with trans-9,10-dihydroxy-9-10-dihydrophenanthrene. The other derivatives were inactive in this test. However, 9-10-dihydroxyphenanthrene and 9,10-phenanthrenequinone were more toxic to the rec- B. subtilis M45 strain than to the rec+ H17 strain. This was also true for phenanthrene 9,10-oxide and 9-hydroxyphenanthrene, but not with the other test compounds that reverted (9,10-dihydroxy-9,10-dihydrophenanthrenes; N-benzyl-phenanthrene 9,10-imine; benzo[a]pyrene 4,5-oxide) or did not revert (phenanthrene, 9,10-bis-(p-chlorophenyl)-phenanthrene 9,10-oxide, 9-10-diacetoxyphenanthrene) the Salmonella tester strains. Although the K region is a main site of metabolism and although all potential K-region metabolites were mutagenic, phenanthrene did not show a mutagenic effect in the presence of mouse-liver microsomes and an NADPH-generating system under standard conditions. However, uhen epoxide hydratase was inhibited, phenanthrene was activated to a mutagen that reverted his- S. typhimurium. This shows that demonstration of the mutagenic activity of metabolites together with the knowledge that a major metabolic route proceeds via these metabolites dose not automatically imply a mutagenic hazard of the mother compound, because the metabolites in question may not accumulate in sufficient quantities and therefore the presence and relative activities of enzymes that control the mutagenically active metabolites are crucial. N-Benzyl-phenanthrene 9.10-imine was mutagenic for the episome-containing S. typhimurium TA98 and TA100 but not for the precursor strains TA1538 and TA1535. This arene imine would therefore be useful as a positive control during routine testing to monitor in the former strains the presence of the episome which is rather easily lost.     

Some Physiological,Microbial, and Toxicological Aspects of the Removal of Phenanthrene by Hydroponic Cultures of Alfalfa (Medicago sativa L.)

Alfalfa (Medicago sativa L.) and other plants bearing an important root system have been shown to be effective in the removal of organic compounds, including polycyclic aromatic hydrocarbons (PAHs). Phenanthrene is one of the main contaminants arising from the petrochemical industry and is included in the USEPA's list of priority toxic pollutants. Hydroponic cultures of alfalfa were employed as a model system to evaluate their capability of removing phenanthrene and to study the plant-pollutant interaction without the interference of a soil matrix. The removal of phenanthrene was followed over a period of 30 days. The half-life of phenanthrene in hydroponics (initial concentration 50 mg L^-1) was reduced 2.7 times when plants were present. Growth index, chlorophyll content of leaves, and peroxidase activity of the roots of plants exposed to phenanthrene were lower than the corresponding values of nonexposed plants. Phenanthrene produced an acute negative effect on the total bacterial counts but also caused an increase in degraders/total bacteria ratio. The Ames Salmonella plate incorporation assay was employed to screen for potential genotoxic metabolites, which could be generated by metabolic activation of the parent compound. None of the samples exhibited a positive response. While lack of a positive response to this test is not a definitive evidence of the absence of genotoxic substances, these results suggest that the plant-assisted removal of phenanthrene merits further investigation.     

Validation of a "clearing" assay for milk lipoprotein lipase in agarose gel

We have developed a simplified method for the quantitative assay of lipoprotein lipase in cow's milk based on the hydrolysis of a glyceride emulsion in semisolid agarose gel. The area of clearing produced thereby is a function of enzyme concentration. Absolute molar rates for unknown samples may be calculated if standards of known activity are used concurrently. The precision of the simplified assay compared favorably with a method based on titrimetric determination of the rate of free fatty acid release. A modified assay has been used to assess the potency of lipoproteins in lipoprotein lipase activation.