JPH06510199A - Detection of toxicity - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] The present invention relates to the detection of toxicity, and more particularly to methods and apparatus for detecting toxicity. Regarding the location.

Background of the invention Generally, toxic compounds are difficult to detect. to detect specific chemicals There are many ways to do this. For example, atomic absorption spectrometry to detect metal ions; X-ray fluorescence, and electrochemical methods, quality for detecting different types of organic substances Quantitative methods, gas chromatography, and ultraviolet absorption to detect a range of compounds There are specific enzyme assays for (see Garbar et al. 1985). However, these The method simply detects whether one compound or one type of compound is present. It's just that. pose a toxicity risk, which is not the goal of these specific tests. If other compounds are present, they remain undetected.

A means to this end is to use biological systems as sensors to detect toxicity. Ru. This allows compounds that contaminate the system to be detected by the sensor.

A range of biological systems can be used to determine whether a system is affected by a toxic agent. A range of methods are used to determine the Early systems used live fish. , fish swimming, catfish movement or electrical activity were monitored. Daphnia survival in water have been used as bioassays (e.g. Dutka and Gowri). a and Gorrie) 1989) a However, these detectors rely on complex, multicellular organisms that are difficult to analyze and difficult to use. It is almost impossible to preserve it in an inactive state for a long time.

Recent developments have shown that single-celled organisms such as algae, fungi or bacteria are common toxicity sensors. (Walker 1988). ). Its advantage is that cells can be easily grown and kept inactive by freezing or lyophilization. It is a small and simple sample that can be stored in This means that it can be easily incorporated into a test system.

The first bacterial haese toxicity assay system was a bacterial mutagenesis test (Maron and Ames (Maron and Ames) 1983), and here suddenly Compounds capable of inducing mutations and initiating malignant transformation It is used to detect bacteria. These assays allow the bacteria to grow further. It relies on creating bacterial colonies that are visible to the naked eye; The number of mutations that occur indicates the number of mutations that have occurred.

More rapid bacterial toxicity sensors detect a range of bacterial, fungal or algal metabolic Depends on the effects of toxins. These effects are measured in a variety of ways. and For example, the MicroTox system uses luminescent bacteria Bulich et al. 1990) was used. Luminescence by these bacteria is due to metabolic Sensitive to reductions in internal sources of energy. Even a small amount of energy production in bacteria Interference will interfere with light emission. This means that the light output will decrease. indicates a potentially toxic substance or interference with intermediate metabolism.

Other assays using single-celled organisms include the uptake of oxygen by microorganisms during their metabolism. or to measure the rate of carbon dioxide production or decrease in pH ( Examples: Karube 1986, Riedel et al. +989 ), any changes in these parameters indicate the presence of toxic compounds. vinegar. Isolated mammalian cells have also been used in this connection. Also, a researcher connects bacterial cells directly to the electrode and determines the size of the general metabolic "suitable temperature". It measures the speed of electron transport. Changes in the electrode potential change the redox state of the bacterial cell membrane. It reflects changes in the metabolic state of living organisms (Turna). Turner) 1989).

Most of these assays must be optimized for specific bacteria.

The susceptibility of specific bacteria varies from toxicant to toxicant, and this determines the sensitivity range of the assay. This means that it can be limited to some substances. Some are technically difficult. accompanied by difficulties.

The purpose of the present invention is to provide a pond approach for toxicity detection.

In one aspect of the invention, a method is provided for determining the toxicity of a sample, the method comprising: contacting the material with living cells and applying waves in the range of 190 nm to 250 nm. and monitoring the absorption of electromagnetic radiation by the cells in the cell.

Single cells are generally transparent to visible light, except for pigments present within the cell. in the cell Due to the presence of proteins and nucleic acids, all cells have a wavelength of 250nm-300nm. Absorbs UV light in the range. Therefore, visible region and 250-300 nm region The absorption of electromagnetic radiation is not useful for immediately analyzing the metabolic status of cells.

However, cells also grow in the 190nm-250nm region (mid-UV of the spectrum). This peak shows an absorption peak in the It will be done. Generally, this peak occurs at approximately 195 nm, but the exact wavelength of the peak is influenced by a wide range of environmental and metabolic influences, as well as toxins, as shown below. It can be done. For convenience, peaks are referred to here as ``peak at N95nm'' or ``peak at min. These expressions are referred to as ``analytical peaks'', and these expressions range from 190 nm to 190 nm, regardless of the actual wavelength. It should be understood that it is intended to cover peaks in the 250 nm range. cell Inhibition of metabolism or rigorous removal of all metabolic substrates and cofactors from the cellular environment This results in a substantial reduction in the absorption of ultraviolet light in the 1l90-250n region. , this is due to two effects. First, the 19 observed for these cells The absorption peak at 5 nm shifts to longer wavelengths, so 1l90-200 The absorption of wavelengths between n and n also decreases. Second, the maximum absorption of this peak is reduced.

The present invention shows that toxins affect cellular metabolism and thus the peak at 195 nm. , based on the fact that it reduces the absorption maximum and also increases the wavelength of the peak. .

The peak at +95 nm is actually due to multiple overlapping absorption peaks. It is. Some toxins affect all of the components in this peak; There are also toxins that affect mushrooms. Details when there is a sample and when there is no sample By monitoring the changes in the absorption peak of the cells, especially the change in the absorption amount at the peak. This provides a powerful indicator of the toxicity of the sample.

This effect is due to the increase in energy within the cell that results in net oxidation in the oxidative phosphorylation pathway. This is thought to be due to interference with lugi metabolism. The reduced heme is It has a substantial absorption peak around 20 Onm, and this heme is oxidized cytochrome heme. This can be seen as a change in absorbance at the analytical peak. Explain. See further discussion below for Tables 1 and 2.

This method can be used qualitatively or quantitatively.

The invention can be used, for example, in batch assays, continuous flow injection analysis, or biosensor applications. It can be carried out in various ways, such as as an essay.

Cells are eukaryotic cells (e.g. protists, algae, fungi, yeast, isolated plant cells, or isolated animal cells, such as cultured mammalian cells), or prokaryotic cells (such as For example, bacteria, mycobacteria, and archaea).

Cells are typically used in assays, even if they are suspected of being toxic. The isolated cells are placed in a dilute aqueous solution containing the sample to be tested for a certain substance. added, at a specific wavelength or multiple wavelengths over the range 1l90-250n. Electromagnetic radiation absorption is measured. Changes in absorption at this wavelength of cells exposed to the sample, or or changes in the absorption pattern at multiple wavelengths compared to cells not exposed to the sample. The magnitude of the toxic effect of the sample can then be determined.

Other wavelengths may also be measured to provide other information.

For example, to estimate the amount of protein or nucleic acid present, The absorption at 280 nm may be measured or the number of cell particles present may be measured. The scattering of light at longer wavelengths (e.g. 600 nm) is measured to estimate You may be

Cells can be directly exposed to potentially toxic samples, e.g. by simple mixing. or the sample may be initially placed in a separate compartment. away from the cells, potentially toxic substances enter the cells via diffusion or osmosis processes. may be reached.

Generally, a detectable response occurs in less than 1 minute after mixing the cells with the sample, typically 15 seconds. Occurs within Reaction time does not seem to vary significantly with toxic concentration .

The method of the invention has been found to be highly sensitive to toxins and is generally is the same or lower concentration that inhibits cell proliferation in conventional assays. It is possible to detect various toxins.

In another aspect of the invention, an apparatus for determining toxicity of a sample is also provided, The device consists of a container containing the sample to be tested and live cells, and a 190nm To monitor the absorption of electromagnetic radiation by cells at wavelengths in the range ~250 nm including the means of

The cells may be present in any convenient form such as. For example, a cell is a suspension of cells. The cells may be present as a liquid, or the cells may be fixed on or in a solid support. For example, cells may be trapped within a polymer matrix or the antibody or other non-antibody so that changes in the cell's absorption spectrum are still detectable. bonded to the surface of the support in a layered manner using a covalent bonding agent or by covalent cross-linking. You may be One convenient embodiment is to pass the sample through and fix it on an agarose membrane. The method includes a flow cell having means for conveniently using the bacterial cells. The sample UV absorption is continuously monitored as it flows through the membrane. Other possible types is a J-type R dipstick.

The apparatus preferably contains two identical containers, such as spectrophotometer cuvettes. one for receiving samples and cells, and the other for control. This is to receive the solution.

The monitoring means preferably includes a spectrophotometer, suitable equipment is commercially available. Ru.

A source for providing radiation (illuminating light) may be provided as required.

A wide range of toxins that affect cellular metabolism can affect analytical peaks. It has been discovered. The present invention therefore provides a broad range of biocides, antibiotics, anti-metabolites. Provides a broad spectrum assay capable of detecting substances and other substances.

The applications to which the present invention can be applied are wide-ranging. The present invention deals with substances that are toxic to cells. Monitor water samples to detect any potentially toxic substances by detecting Examples of water samples include rivers, soil , drinking water supplies, mines, industrial wastewater such as large-scale metal processing and paper manufacturing, agricultural or This includes household wastewater. The present invention can be used, for example, with air (not vented by passing air through water). Toxins from other environments such as from dry surfaces or solids (by any number of collection methods) to detect the presence of toxic substances in the water used to collect them. It may be used in the same way. The invention also applies to industries such as hotels, hospitals, restaurants, etc. It can also be applied to quality control of water used on the consumption side.

The present invention also utilizes known techniques by exposing cells to substances and monitoring the results. It may also be used to screen for biocidal activity, etc. by measuring the toxicity of the substance.

The invention also provides for use in monitoring oxidative phosphorylation processes in living cells. I can do it.

The invention will be further explained by way of example in the following examples with reference to the accompanying drawings. , Figure IA shows different concentrations for Bacillus subtilis cells. 2 is a graph of absorbance versus wavelength showing the effect of the toxin 2-mercaptoethanol on , Figure IB is a graph of absorbance versus wavelength showing the effect of washing out micronutrients from Bacillus subtilis. and Figure 2A shows the effects of incubating B. subtilis cells with varying concentrations of sodium azide. ○ indicates the OD at the peak, and ・ indicates the change in absorbance at 200 nm. A graph of optical density (absorbance) versus concentration of sodium azide (ppm) showing the OD of It is f, Figure 2B shows the effects of incubating Bacillus subtilis cells with varying concentrations of sodium azide. , the effect on the wavelength of the optical density (OD) peak between 190 nm and 250 nm. A graph of analytical peak wavelength (nm) versus sodium azide concentration (ppm) showing It is f, Figure 2C shows the effect of different concentrations of sodium azide on the growth of Bacillus subtilis cells. , is a graph of reciprocal time (hours) versus concentration of sodium azide (ppm), and FIG. 3A is the change in OD200nm of Bacillus subtilis exposed to different chemicals at various concentrations Normalized OD (OD200nm 10D260nm) vs. concentration (uM ), O indicates the results for iodoacetic acid, and . indicates the results for oxamic acid. △ indicates the results for trimethoprim, FIG. 3B is a graph similar to FIG. 3A, where . indicates results for silver nitrate, and O indicates the results for sulfamethazine. However, FIG. 3C is a graph similar to FIG. 3A, and * indicates the results for glucose. and △ indicates the result for sodium chloride, FIG. 4 is a graph similar to FIG. evisiae) and show the effect of five different concentrations of sodium azide on Figure 5 shows the OD of Satucharomyces cerevisiae exposed to different toxins at various concentrations. There is a graph of optical density versus concentration (ppm) showing the change in sodium acetate. shows the results for glucose, mouth shows the results for glucose, and mouth shows the results for anodized sodium. FIG. 6 shows a cell of an example of an apparatus for carrying out the present invention. and FIG. 7 shows an apparatus incorporating the cell shown in FIG. Figure 8 shows Tween-20 (Figure 8a) and sodium azide. A graph of OD vs. time at 200 nm showing the results obtained using It is f.

Example Bacterial cells were obtained from NCIMB (1--Re Research, Aberdeen, Scotstran). Torrey Research, Aberdeen, Sc.

L-broth (1% fermented) was obtained from Mother extract, 05% Bacto l-ributone, and oxoite. Chemicals (Oxoid Chemicals Ltd.) to manpower, and 1 96 sodium chloride). 1ml or 100ml of overnight bacterial culture ml of broth and inoculated into bacteria until a convenient density is reached, usually 60 min. Optical density (OD) at 0 nm or 0. It is cultured until it reaches between 1 and 0°3. Ta. The bacteria were grown at 4°C.

rvall) by centrifugation at 6000 rpm using an RC-5B centrifuge. and resuspended in water for use. Bacteria in this preparation are kept at room temperature. There were enough micronutrients present to keep it "non-toxic" for several hours.

S. cerechisiae culture is a commercially available freeze-dried brewer's ferment. Prepared from mother (Boots p.c., UK) in L-bouillon. cultured and processed as described above. Other chemicals are from the UK's Sigma Obtained from Sigma Chemical Co., Ltd.

Two quartz spectrophotometer cuvettes with a path length of 1 cm were prepared, each of the cuvettes contained 1 ml of distilled water. On the one hand, the preferred OD at 200 nm, Typically, amounts of bacterial cells ranging from 1 to 2 were added. both sides of cuhette A potentially toxic dilute aqueous solution was added to the drug. Solution in both cuchettes Added 190nm-250nm for potentially toxic solutions. This is because the absorbance is large in the m range. Second cuvette without bacterial cells is a “blank” control cell, against which cuvettes containing bacteria are Optical density is measured. OD from 190 to 350 nm is available from Phil 5P8-500 UV/visible scanning spectrometer. 260nr The CD at n or 280 nm is considered a measure of the total amount of protein involved. , the OD at 350 nm was considered a "Haesline" absorption. these two The value of is a control value and can be used if necessary, e.g. In order to compensate for the slight change in the number of Used to normalize other measurement results.

Various different experimental results are shown in the figure.

Figure IA shows different amounts of 2-mercaptoethanol, a known protein denaturing toxin. The effect on the spectrum when added to cells of Bacillus subtilis strain 8054 is shown. Tan The absorbance at 260 nm by protein and RNA remains virtually constant. However, the analytical peak at approximately 195 nm changes significantly, i.e. the wavelength of the peak increases and the peak height decreases. A similar pattern was observed using a large number of compounds. It can also be obtained in widely varying concentrations.

Figure IB shows the effect of washing micronutrients out of the cell, showing that low levels of The poison has a similar effect on washing cells, or the effect of high concentrations of poison is on washing cells. It shows that the effect is different in quality.

Figures 2Δ, 2B and 2C show a wide spectrum of biocides known as acyl. The results of a pond experiment using 1 helium are shown. Figure 2A shows the added reed OD at 200 nm and 195 nm peak plotted for the amount of OD at the top of the peak (the top of the peak may be at the top). Figure 2B is It shows the wavelength of the absorption peak between 190 nm and 250 nm, also added It is plotted against the amount of thorium anodide. Figure 20 shows the results for these cells. This figure shows the biocidal effect of anodized 1-Rium. Cells are anodized in different amounts)・Rium Their OD at 600n, m or iU! I was determined , which gives the magnitude of the cell density. Figure 20 shows different concentrations of cells in the growth medium. The reciprocal double time of cells during the exponential growth phase for lithium azide (this is the cell is the magnitude of the growth rate). Compare Figures 2A, 2B and 2C. In summary, the technology of the present invention shows that the same It has been shown that the concentration of sodium chloride can be detected, which means that the response of this technology is tion is correlated with the proven biological effects of lithium azide. It is.

Figures 3A and 3B show the range of pond biocides further detected by the techniques of the present invention. shows. Figure 3A shows the technology of the present invention, trimethoprim (a specific antibiotic), and and oxamic acid and iodoacetic acid (these are widely used because they inhibit certain steps of metabolism). demonstrated to detect a spectrum of biocides (acting as biocides). Figure 3 B is the technology of the present invention or the use of sulfamethacin (other specific antibiotics) and silver nitrate (heavily is a metal salt that inactivates many enzymes in many biological systems). show.

In contrast, Figure 30 shows that this sensor is a compound that is not toxic to this organism. , shown to be unaffected by high concentrations of glucose or sodium chloride. vinegar. Therefore, the technology of the present invention is unique in distinguishing between toxic and non-toxic substances. It can be said that he is highly sensitive and highly sensitive.

Tables 1 and 2 rank the optical effects of ultraviolet light on metabolism and toxins. -Summary of the influence of some compounds on the ultraviolet spectrum. three EC50 measurements are used; these are the absorbance at 200 nm (the most sensitive high measurement), the absorbance of the absorption peak from 190 nm to 260 nm, and its peak The effect of various compounds on the wavelength of the peak is addressed. (EC50 is one The change from the parameter's "control" value is 50% of the maximum possible change. The concentration is as follows. But what if sodium azide is OD? . from 2 to 0 If you descend to , EC50 is OD. . The concentration is such that it becomes l. )(value Some of these are not filled in, or this may indicate the range of concentrations tested or their values. (usually this is due to the marginal effect of solubility). (It is for the sake of fruit.) ) Table 1 shows the effects for a range of metabolites, with EC50 They are arranged in order of decreasing value. In particular, this table also lists compounds with their oxidation The relative ratios of hydrocarbons to oxygen are shown in column 6 of the table. It will be done. This table shows that reduced metabolites, especially sugar, have little effect on Bacillus subtilis' UV absorption. This shows that there is no impact. Metabolites that are more oxidized have more dramatic effects . This indicates that there is a link between the metabolic outcome of a compound and its effect on UV absorption. Indicates that there is some stiffness.

Table 2 lists EC50 values for several metabolic inhibitors. center Some inhibitors of human metabolism also lower the absorbance around 20 Onm and Concentrations similar to those known to inhibit cell metabolism and growth , which is much lower than the concentration of metabolite required to show the same effect. It shows the effect of One substantial exception is sodium cyanide. different concentrations of cells exposed to sodium cyanide at the same concentration of cyanide. Compared to the reverse growth rate of cells that have been It has been shown that this metabolic inhibitor occurs at much higher concentrations than the effects on Ru.

Table 3A summarizes the effect of a number of antimicrobial agents on the bioassay of the present invention. be. This assay tests for compounds that inhibit bacterial growth or metabolism through several pathways. It sensitively detects organic compounds. In contrast, its response to heavy metal toxicants occurs at higher concentrations. Table 3B shows equivalent measurement results for non-toxic salt solutions. However, this is due to the specific effect of the toxin on cells, and the The general effect of loaded heel or metal counter ions is not show. Sodium bromide is an exception (potassium bromide is not listed but has the same effect) ), which has significant spectroscopic effects at around 1 mM.

Spectral effects of compounds on other cell types The observed spectral changes are Therefore, they occur against one of the prominent chromophore components of Bacillus subtilis cells. It must be. This may be a cellular component specific to Bacillus subtilis, or it may be present in many types of organisms. It can be a common component of things. The general effect of compounds on mid-UV absorbance is Two other organisms were analyzed to see what they were like.

Both E. coli and S. cerevisiae are The same intermediate-UV absorbance changes occur when exposed to substances that are likely to alter the next-order metabolism. to show the

The optical effects on Satucharomyces cells are shown in Figures 4 and 5. yeast in water The spectrum of cells differs from that of bacterial cells, especially if different concentrations of cell suspensions are used. If the temperature is adjusted so that the absorbance at 260 nm is the same, yeast cells The scattering of light by the bacterial cells is substantially greater than that by the bacterial cells, and at 195 nm The relative height of the absorbance peaks is higher for yeast cells than for bacterial cells. becomes larger. However, if these cells are exposed to metabolites or toxins , characteristic changes similar to those seen in Bacillus subtilis cells are observed.

Figure 4 shows the results of incubating Satucharomyces cerevisiae with sodium azide. This shows the change in the spectrum of the fruit. When the concentration of sodium azide is increased, it becomes 190 nm. The height of the absorbance peak between 260 nm and 260 nm is reduced, and the peak wave of that peak lengthen or increase.

Figure 5 shows the effects of two typical metabolites on these cells and shows the effects of intermediate-purple The effect on the external spectrum is very similar to that of the same compound on B. subtilis cells. Show that there is. The effects of sodium acetate also affect these organisms, including Bacillus subtilis. (compare Table 1), but yeast cells are more susceptible to bacteria than Bacillus subtilis. It can be seen that there is little reaction to the course. This is probably because this yeast strain is used for brewing. therefore, for the high concentrations used here, normal immersion This is because B. subtilis and the Bacillus subtilis shown here are The effects of glucose on E-C01i and E-C01i indicate global cellular damage due to osmotic effects. It is something.

Generally, experiments using E-Coli cells and mammalian cells in culture are similar. Similar results (not shown) were obtained.

Figures 6 and 7 show flow cell configurations a"c suitable equipment for carrying out the method of the invention. An example of this is shown below.

FIG. 6 shows the basic cell configuration. The quartz cuvette lO has a clip 12 inside it. By supporting the agaro membrane 14 with a low gelling temperature, the bacterial test cells are placed in a normal manner. is fixed in it. A silicone stopper 16 is placed on top of the cuvette and Each tube 18.20 carries liquid into or out of the cuvette. flow, so that the test sample can flow through the membrane in the direction of arrow 22. Ru.

Figure 7 shows the assembly of the flow cell. Two cell configurations shown in Figure 6 A suitable spectrophotometer, such as the Philips 5P8-500UV/visible spectrophotometer ( (not shown). The test cell 24 contains a membrane with bacterial cells, while the Control cell 26 includes a membrane free of bacterial cells.

In use, a sample to be tested is pumped from container 30 by pump 28. the test cells and controls through their respective tubes 32, 34. It is carried to the inlet of the cell, flows through the membrane and exits the cell into the container 36.

The spectrophotometer measures the absorbance of the two cells at 200 nm as the sample passes through the membrane. Compare.

Figure 8 shows a typical recording line obtained from an experiment using the apparatus of Figure 7, with 0.01% Exposure to Tween 20 (Figure 8a) and 10 ppm thorium azide (Figure 8b) OD is plotted at 200 nm versus time during this period. these According to the figures, the sensor reacts to the poison in about 10 seconds, and it takes 1-2 minutes to cause near-fatal poisoning. recover from. If the sensor has not been exposed to extremely toxic substances, at least It has an operating life of 11 hours (the longest time tested so far). do.

Consideration - Examples listed below include Bacillus subtilis, E-Coli and S. cerechis The UV absorption properties of all cells are highly influenced by metabolites added to their cells. Indicates that you will receive. This is the original The effects of compounds on basic metabolic processes common to karyotes and eukaryotes must differ. That is to say, yes. Changes detected by mid-UV spectroscopy indicate that the compound is prokaryotes that affect cells within 15 seconds of incubation and are grown under aerobic conditions effects on organisms and eukaryotes. They are therefore responsible for (DNA replication or for central metabolism (rather than slower processes such as cell division) and for prokaryotic There must be an effect that the compound has on aspects of metabolism common to organisms and eukaryotes.

The effects of surfactant compounds such as Tween-20 and their interference with ion transport functions. The effects of substances such as sodium uride can be seen as changes in the mid-ultraviolet spectrum. The effect detected is closely related to membrane proteins, membrane ion partitioning, or both. This indicates that this is a change in the relevant metabolic aspect.

The above results indicate that some of the oxidative phosphorylation (OP) enzymes in these cells It has been shown that the redox state is consistent with that detected by mid-UV spectroscopy. be suggested.

These enzymes charge-transfer iron atoms to their heme moieties. -transfer) very broad mid-ultraviolet caused by absorption bands It is expected that it will have an absorption hunt. The "normal" state of high absorbance is reduced The poisoned J state, which corresponds to heme and has low absorbance, is said to correspond to oxidized heme. It is suggested that. In actively growing cells, OP chains are initially reduced. It is in a state of If intermediate metabolism is inhibited, the supply of something such as reduction to the OP chain When blocked, all components of the OP chain are rapidly oxidized due to the continued activity of chain enzymes. and decrease mid-UV absorbance. Similarly, already as the sole carbon source By providing cells with partially oxidized metabolites, reducing equivalents can be converted into oxidized energy. It takes away from ghee metabolism and begins to assimilate its compounds. Compounds are more oxidized Then, more reducing equivalents are required. Relatively reduced glucose etc. Sugars instead generate reducing equivalents during metabolism, thus providing reducing equivalents to the OP chain. “Feed” things.

In this scheme, the anomalous effects of sodium cyanide on cells are Waited. Among the metabolic inhibitors listed in Table 2, cyanide is an enzyme at the end of the OP chain. Inhibits certain cytochrome C oxidase. This effectively inhibits cell proliferation. However, sodium cyanide has little effect on the mid-UV absorbance of cells. stomach. If the mid-UV absorbance is characteristic of the redox state of the OP chain, then Or expected. In other words, inhibiting sotochrome C oxidase inhibits the OP chain. It also inhibits the oxidation of the OP chain enzyme, thus causing the oxidation of the OP chain enzyme. do not have.

This study shows that the ultraviolet absorption spectra of bacterial and yeast cells are affected by various metabolites and We show that this can be manipulated by adding metabolic inhibitors. monitor It is thought that the absorption characteristics of It's appropriate. Whether this mechanism is correct or not, this Elephants are classified into bacteria and how they react to numerous metabolites and metabolic inhibitors. It can be useful for If this mechanism is correct, it depends on where you pay attention. Oxidative Phosphorase Oxidation in Living Microorganisms Using Simple and Standard Laboratory Equipment It can provide a simple analytical tool to observe the reduction state.

Double'V: Margin Table 1 Effect of metabolites on the mid-UV spectrum of Bacillus subtilis cells Column 1: Metabolites tested. Columns 2 to 4: Evaluate the effect of metabolites on cells EC50 for each metabolite using different spectral parameters to value. Parameters are: 2nd column: OD at 200 nm, 3rd column: 26 from 190 nm OD at absorption peak at 0 nm, 4th column = absorption peak from 190 nm to 260 nm wavelength. Column 5 Oxidation rate of 1 metabolite R0C = number of carbon atoms, H = number of hydrogen atoms, 0 = Number of oxygen atoms, then R = (4XC+H)10゜*: Oxamic acid is in is an inhibitor of tricarboxylic acid cycle enzymes isolated in vitro, but in Bacillus subtilis cells. It does not significantly inhibit growth (results not shown) and is therefore considered here as a metabolite. do.

Glucose 30000 30000 - 6 Fructose 9000 200 00 - 6 Succinic acid 1500 1600 1600 5.5 Sodium acetate 800 8000 1000 5.5 Citric acid 400 1500 650 4.57 Malic acid 400 400 1000 4.4 Oxamic acid *12 4 0 35 3.3 Table 2 Metabolic toxins for mid-ultraviolet spectrum of Bacillus subtilis cells effect.

Column 1 1 metabolic toxins. Columns 2-4: Different variables used to assess the effect of metabolites on cells. EC50 value for each metabolite using spectral parameters. Parameter Data: 2nd row: OD at 200nm, 3rd row: absorption from 190nm to 260nm OD at peak, 4th column: wavelength of absorption peak from 190 nm to 260 nm. *Thin The sensitivity of cells to silver nitrate is due to residual chlorine in the medium rendering the silver relatively inert. It is highly dependent on the amount of culture medium left in the cell preparation for precipitation.

Sodium azide 15 25 20 Silver nitrate* 10 2 15 Iodoacetic acid 42 50 50 Chloramphenicol 50 120 80 Sulfamethacin 16 20 2 0 Trimethoprim 27 50 50 Table 3A 2-mercaptoethanol 1.53 1.91 1.90 Chlorinated steel (1) 0. 10 0.10 0.10 Copper sulfate (11) 1.58 1.58 ND iodized vinegar Acid 0.22 0.26 0.20 Kanamycin 0.32 0.34 0.3 Mercury tetrachloride (TI) 0.34 0.68 0.40 Nalidixic acid 0.086 0.086 0.172 Sodium bisulfate 0.86 ND f), 96 tri Metobrim 0.093 0.17 0.20 Table 3B Cesium chloride 100 or more 100 or more Fructose 47 10 0 or more, 100 or more glucose, 160 or more, 160 or more, 160 or more inosites Rule 50 '65 65 N-acetyl 25 25 30 glucosamine Potassium chloride 30 or more 30 or more Sodium bromide 0.5 1 1 Sodium fluoride 20 20 25 Trizma 20 25 25 (Trishydroxymethyl) Explanation of Table 3 Concentration of compound that caused a half-maximal change in the spectral properties of Bacillus subtilis cells (mM). Column 1, compounds. Columns 2 to 4: Causes a change of half the maximum value concentration.

4 houses ( Bulich, A.A., Tung, K.-K. and 5cheiber, G. (1990), Journal of Bioluminescence an d Chemilu+n1nescence 5. 71-77゜Dutka, BJ and Gorrie, JF (1989), Enviror+me ntalPollution　57,1. -7゜Garbgrino, JR , Steinheimar, T. Rand Taylor, B. E. (19 85).

Analytical Chemistry 57.46R-88R.

Karube, I (1986), -Funda+++eoea1g and Applicaeior++; ofChe! ll1cal 5ensors- , pp330-348゜Maron, DM and Awes, BN (]-983), Mutation Re5earch 113°173-21 5゜ Re1del, K., Renneberg, R. Wollenberger, 11. Kaiser, G. and Scheller, F.W. (1989) , Journal of Chemical Technology andB iotechnology 44.85-106゜Turner, A　P　F　 (1989), 5 sensors and actuators 17 433- 450°Walker, JD (1988), Journal WPCF 6 0,11.06-1121゜11000pp 500ppm 400ppm 2 00pprn n. 2-MEY Zo4 → -1-11 Ushi Geo [Month L [ppm l0.1 1 10 100 1000 ? ＞'A IL r) Two 1ft-Pity (ppml(LOI □-1'+ 10 100 1000 10000 Epu Plant T Hiso I7 Muzu1 Hat [p Rabbit) Q ,001 0.01 0.1 1 100.0OOOO10,00010,001 0,010,111010010000.01 0.1 1 10 100 1 000 international search report 10-N---Mu-11w1lss N&PC/GB 92101618 International Investigation report Sum*+y:;=b-Shini:”*+w”Z’een　e’em　’l°ly　 7m::;;, 2;=-;@, +s;fuko, 7S7;? , VHv; ten e H*m　Is　lh++　m1°”aMt17!Sel1msl　mmmh　 -a↑M*@jlowampPal・-1elntmIIIRsa豐vyllsk l*jar he 嘗-ulllcvnrtwhich*r++f weir | 11 辂y @1yesle+Ihyu-chi1-hikaisll+or++y+1ms.

Continuation of front page (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, CB, GR, IE, IT, LU, MC, NL, SE), 0A (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, SN, TD, TG), AT, AU, BB, BG, BR, CA, CH, C3゜DE, DK, ES, FI, GB, HU, JP, KP, KR, LK, LU, MG, MN, NiW, NL, NO, PL, RO, RU, SD, SE, US

Claims (13)

[Claims] 1. contacting the sample with living cells and 190 nm to 250 nm waves; monitoring the absorption of electromagnetic radiation by the cells over a range of Methods for determining toxicity. 2. A step that monitors the amount of light absorption in the wavelength range from 190 nm to 250 nm. 2. The method of claim 1, comprising: 3. Cells may be bacterial cells, yeast cells, or cultured mammalian or plant cells. 3. The method according to claim 1 or 2, comprising: 4. Claim 1, 2 or 3 further comprising the step of monitoring absorption at other wavelengths. or the method described in 3. 5. A method according to any of the preceding claims, wherein the cells are present as a suspension of cells. . 6. Any of claims 1 to 4, wherein the cells are immobilized on or in a solid support. The method described in 7. A container for holding the sample to be tested and live cells, and a 190nm means for monitoring the absorption of electromagnetic radiation by cells in the wavelength range from 250 nm to 250 nm. and an apparatus for determining the toxicity of a sample. 8. one for the sample and cells and the other for the control solution. 8. The device of claim 7, comprising two identical containers for storing. 9. A claim comprising a flow cell having means for passing a sample therethrough. The device according to item 7 or 8. 10. Device according to claim 7, 8 or 9, wherein the cells are present as a suspension of cells. . 11. According to claim 7, 8 or 9, the cells are immobilized in or on a solid support. The device described. 12. 12. The means for monitoring comprises a spectrophotometer. equipment. 13. 13. The method according to any of claims 7 to 12, further comprising a source for providing radiation. equipment.