WO2004033714A1

WO2004033714A1 - Identification and modulation of intermediates of alcohol metabolism

Info

Publication number: WO2004033714A1
Application number: PCT/IL2003/000812
Authority: WO
Inventors: Melvyn Rosenberg-Nevo; Amir SHUSTER
Original assignee: Ramot At Tel-Aviv University Ltd.
Priority date: 2002-10-10
Filing date: 2003-10-09
Publication date: 2004-04-22
Also published as: IL152219A0; AU2003269463A1

Abstract

The present invention discloses a method for determining metabolism of a metabolite, comprising the steps: a) adding a biological sample adjacent to or upon a matrix, said matrix comprising immobilized blood cells or a blood substitute; b) incubating said matrix in the presence of said metabolite; c) assessing the degree of hemolysis occurring in said matrix, wherein a large degree of hemolysis is equated with a high degree of metabolism of said metabolite. In a preferred embodiment, alcohol is the metabolite utilized. The invention further discloses a kit for measuring the ability of a sample, to metabolize alcohol. Also disclosed is a method for determining whether a subject has a high risk of developing cancer. Further disclosed are a method for evaluating the cytotoxicity of an entity, and a method for evaluating the ability of matter to prevent cytotoxicity.

Description

IDENTIFICATION AND MODULATION OF INTERMEDIATES OF ALCOHOL METABOLISM

FIELD OF THE INVENTION

The present invention relates to alcohol metabolism and to harmful intermediates, such as acetaldehyde, of alcohol metabolism. More specifically, the invention relates to a method for measuring the ability of microorganisms, to metabolize alcohol. The invention further discloses a method for screening a human for the risk of developing cancer, especially oral and pharyngeal cancer. The invention further discloses a method for screening cells or tissue for changes in metabolic patterns. The invention further discloses a method for assessing the ability of living cells to degrade toxic substances, namely their ability to perform bioremediation. The present invention further relates to the identification of cytotoxic agents, and to a method of appraising the capacity of other agents to modulate these cytotoxic agents.

BACKGROUND OF THE INVENTION

The environment is replete with toxic agents that affect the health and wellbeing of ecology and human health. Foods, cosmetics and other products proximal to humans may contain harmful toxic agents. There is currently much interest in determining new methods for observing the potential damage of various agents and treatments and in finding ways to mitigate such damage. Conversely, in other situations (antisepsis, fighting cancer), novel agents and treatments are constantly being sought out, that can harm specific cells and tissues.

Most of the tests available to check for toxicity are complicated, and some involve harming animals. The inventors have found a method, described below, which enables simple and rapid observation of the harmful effects of certain alcohols and their byproducts, and mitigation of such effects. Although the inventors have concentrated on alcohols, there is no intention to limit the scope of the invention to alcohols alone. The method for assessing cytotoxicity can be used as a general procedure to test for potential harmful effects, and to test the ability of other agents to remediate these harmful effects.

Microorganisms that grow in the presence of alcohols, particularly ethanol, have great industrial importance. From an industrial point of view, wine and brewery fermentation is based on alcohol production by microorganisms, often the yeast Saccharomyces cerevisiae. Such microorganisms produce alcohol from sugar, yet despite the inhibitory effects of the alcohol, continue to produce alcohol as its concentration rises in the fermenting liquid. High tolerance of ethanol is one of the most desirable characteristics of wine yeast, alongside rapid initiation of fermentation, high fermentation efficiency, high osmotolerance, low temperature optimum and moderate biomass production, all of which are related to ethanol metabolism. Approximately 250 genes are associated with ethanol tolerance in yeast; however, researchers have not yet succeeded in significantly increasing the ethanol tolerance of yeast. The development of yeast strains with increased resistance to high ethanol concentrations remains one of the major challenges of the wine industry. The need exists for a method that allows rapid detection and isolation of microorganisms having a high ethanol tolerance.

Alcohol metabolism is one example, illustrated above of the metabolism of molecules with applications in the areas of industrial biotechnology and health. Aerobic degradation of alcohols is related to competent mitochondrial function, DNA repair mechanisms, iron uptake and metabolism, oxidation and reduction processes, stress, oxygen detoxification, membrane integrity, and cell-cell interactions. These mechanisms are common to a wide variety of metabolic processes. The need exists for a method that allows rapid detection and isolation of microorganisms and tissues with altered metabolic functions.

Oral and pharyngeal (throat) cancer are the sixth leading cause of cancer in the U.S., accounting for some 30,000 new cases each year. This type of cancer is particularly difficult to treat. Both alcohol consumption and smoking are important risk factors for oral and pharyngeal (throat) cancer. The carcinogenic effects of ethanol are unclear. In contrast to smoking, which creates a variety of hazardous compounds, ethanol itself is not carcinogenic. Acetaldehyde, the first metabolite of ethanol, however, is carcinogenic and it is likely that this by-product is the major culprit. Interestingly, acetaldehyde is also considered a potential carcinogen in tobacco smoke. In classic ethyl alcohol metabolism, ethanol is absorbed into the stomach or the small intestines and transferred to the liver through blood vessels. Alcohol dehydrogenases, present in liver and stomach cells, catalyze the oxidation of ethanol to acetaldehyde, which in turn is decomposed into acetic acids by acetaldehyde dehydrogenase (ALDH) in the liver cells. These resultant acetic acids are eventually converted to CO and H O in muscle and adipose tissues.

U.S. Pat. No. 6,120,806 discloses use of cyanamide (in controlled-release form) as an alcohol-deterrent, which is thought to act through inhibition of hepatic acetaldehyde dehydrogenase enzymes (ALDH). When alcohol is then consumed by the subject, acetaldehyde accumulates at high concentrations, causing intense sickness in the subject. This intense sickness associates negative feedback with alcohol consumption, thereby conditioning alcohol-drinkers to become alcohol-averse.

During alcohol metabolism, alcohol dehydrogenases present in the human mucosa form acetaldehyde. Oral and pharyngeal microorganisms contain active alcohol dehydrogenases as well. It has recently been postulated that bacteria and yeast present in the mouth, tliroat and other areas of the gastrointestinal tract, may increase one's risk of developing oral, pharyngeal and gastrointestinal cancer. These natural flora metabolize alcohol by converting it to the carcinogenic substrate acetaldehyde. Recent studies by Homann et al. (Carcinogenesis, 18 (9), 1739-1743, 1997; Carcinogenesis 21 (4), 663-668, 2000) have shown that ingestion of moderate amounts of ethanol leads to production of marked amounts of acetaldehyde in saliva. Considerable fluctuations in acetaldehyde production capacity were found in comparing the saliva of different individuals; gram- positive bacteria and yeasts were associated with higher acetaldehyde production. The enzyme alcohol dehydrogenase is present in widely varying amounts among the various microorganisms that make up the oral flora. Microorganisms with high levels of alcohol dehydrogenase are more prevalent in heavy drinkers. This may explain the predominance of oral and throat cancer among heavy drinkers. Additional findings showed that acetaldehyde formation in vivo was inhibited upon rinsing with an antiseptic mouthwash (chlorhexidine), which apparently reduced microbial alcohol-metabolizing activity. Smoking and heavy drinking were the strongest factors increasing salivary microbial acetaldehyde production among 326 volunteers. The need exists for a method for screening humans for the risk of developing cancers related to alcohol metabolism, especially oral, pharyngeal and gastrointestinal cancer.

Aldehyde production by microorganisms is measured using gas chromatographic determination (for instance, of acetaldehyde), a complex procedure; or using an assay that follows the reduction of NAD+, by means of UV spectrophotometry. Both these procedures are laborious and time consuming. An agar indicator plate for detection of bacteria that produce aldehydes is described by Conway et al. (Journal of Bacteriology, 1987, vol. 169: 2591-2597). In that article, an agar is used that can undergo a color change if acetaldehyde is generated; the acetaldehyde oxidizes pararosaniline (Schiff reagent) present in the agar, causing a color change. However, this agar suffers from many drawbacks: it is toxic and not amenable to widespread microbial growth. The agar is sensitive to light, smoke, plastic containers and temperature, and must be prepared the same day of use, since it cannot be kept for longer than two days. During use, the color diffuses away from the colony, making it difficult to judge the intensity of the reaction. In our laboratory, yeasts with powerful acetaldehyde-generating potential appeared negative (e.g., Candida krusei).

The need exists for a successful method that allows direct isolation, enumeration and identification of alcohol-metabolizing microorganisms from samples. Applications for such a method would include, for instance, examination of the degree of alcohol metabolism of saliva samples for research purposes, or in order to screen individuals for the risk of developing oral, pharyngeal or digestive tract cancer. An additional application is the isolation of microbial strains that are of interest to the wine and brewery fields.

It is the object of the present invention to provide a facile method and kit for measuring the ability of a sample, to metabolize alcohol. The method is simple and rapid, and is based on a colorimetric assay.

It is the object of the present invention is to provide a method for screening individuals for the risk of developing cancer, especially useful for evaluating the risk of developing oral, pharyngeal or digestive tract cancer.

It is the object of the present invention to provide a facile method for evaluating the cytotoxicity of an entity, and another method for evaluating the ability of matter to prevent cytotoxicity.

It is the object of the present invention to provide a facile method for evaluating the metabolic characteristics of an entity

These and other objects of the present invention will become more apparent in view of the summary and the detailed description of the invention that follow.

GLOSSARY

In the context of the present invention, the term "alcohol metabolism" and the like, refers to biological phenomena occurring in the presence of alcohol, that can be detected using the disclosed assay, including but not limited to the conversion of alcohol to aldehyde and to other toxic molecules.

In the context of the present invention, the term "biological sample" refers to any sample comprising active biological molecules, which are present either in suspension or are enclosed within a eukaryotic or prokaryotic cell.

In the context of the present invention, the term "bioremediation" refers to the process by which living organisms act to degrade or transform hazardous organic contaminants into non-toxic materials.

In the context of the present invention, the term "blood substitute" refers to a colored material encapsulated, such as by a membrane or by a liposome, or to compartmentalized hemoglobin, such as hemoglobin encapsulated by a membrane or by a liposome.

In the context of the present invention, the term "cytotoxicity" refers to the degree of harm caused to living tissue, as measured by the relative degree of hemolysis of immobilized red blood cells.

In the context of the present invention, the term "entity" refers to a material that is undergoing evaluation in order to assess the degree of cytotoxicity it possesses. Examples of such entities could be chemical reagents, radiation, biological tissues, individual biological cells, or aldehyde-producing cells.

In the context of the present invention, the term "matter" refers to a material undergoing evaluation to assess its ability to prevent the effect of a cytotoxic agent. It may thus be considered an anti-cytotoxic agent. Examples of such entities could be chemical reagents, biological tissues, or individual biological cells, for instance yeast, fungi, bacteria, human or plant cells.

SUMMARY OF THE INVENTION

The inventors have discovered that certain microbial colonies that are capable of alcohol metabolism, can be identified as such when they are grown on blood agar plates in the presence of alcohol. The inventors disclose that a halo of hemolysis can be seen around colonies which metabolize alcohol. In the absence of alcohol, no hemolysis is observed around these colonies, or the appearance of the hemolysis is dramatically delayed. The inventors acknowledge that it is common microbiology technique to identify microorganisms by plating samples on a blood agar plate, in order to distinguish them based on their endogenous hemolysis, or based on joint hemolysis in the presence of certain other microorganisms. However, microorganisms that were not previously seen to be hemolytic, were surprisingly discovered to be hemolytic when grown in the presence of alcohol. Most of the microorganisms of importance in alcohol metabolism, both in the body and in the fermentation industry, are not hemolytic unless they are grown in presence of alcohol.

The above discovery has several applications disclosed hereby. This plating method can be used for research purposes, and for isolation of microbial strains exhibiting different hemolytic patterns, which may be useful for the wine and beer industries. Additionally, microorganisms which are capable of alcohol metabolism can be identified in order to screen humans for the risk of developing cancer, especially oral, pharyngeal or gastrointestinal cancer. Individuals having a high quantity of microorganisms with extensive alcohol metabolizing abilities may be at higher risk of developing cancer. An additional disclosure of the present invention is the use of hemolysis as a visual gauge for evaluation of the degree of toxicity towards biological cells, which is present, for instance, in a novel compound. Any entity being studied to determine if it is toxic for humans or animals can be brought into contact with immobilized red blood cells, and toxic substances may lyse the red blood cells, creating a halo around the blood cells, which is easily seen. In addition, matter being evaluated to determine if it provides protection from a known toxin, can be brought in contact with the toxin and with immobilized red blood cells, in order to see the material grants "protection" from the toxin, namely if the hemolysis is averted.

There is thus provided in the present invention, a method for determining metabolism of a given metabolite, comprising the steps of: a) adding a biological sample adjacent to or upon a matrix, said matrix comprising immobilized blood cells or a blood substitute; b) incubating said matrix in the presence of said metabolite; c) assessing the degree of hemolysis occurring in said matrix, wherein a large degree of hemolysis is equated with a high degree of metabolism of said metabolite, and a small degree of hemolysis is equated with a low degree of metabolism of said metabolite.

In one preferred embodiment, the biological sample comprises microorganisms. In another preferred embodiment, the metabolite is added in vapor form.

Moreover, according to a preferred embodiment, said blood cells are aged to sensitize said blood cells to hemolysis.

Additionally, according to certain embodiments, said matrix is a semi-solid.

Moreover, according to a preferred embodiment, the matrix is a selected from the group consisting of: agar, gelatin, cellulose, methylcellulose, nylon, paper, glass wool, alginate, guar gum, xanthan and carboxymethylcellulose.

In a preferred embodiment, the method identifies and measures the ability of a sample, to metabolize or modify alcohol, using the steps of: adding said sample to a matrix, or adjacent to a matrix, wherein said matrix comprises immobilized blood cells or an immobilized blood substitute; adding alcohol above, onto or adjacent to said matrix; incubating said matrix; ascertaining the degree of hemolysis occurring in said matrix. A large degree of hemolysis is equated with high alcohol metabolism abilities, and a small degree of hemolysis is equated with low alcohol metabolism abilities.

In one preferred embodiment, the biological sample comprises microorganisms.

Additionally, according to a preferred embodiment, the alcohol is added in vapor form. In such case, in a most preferred embodiment, alcohol vapors originate from a wickable material placed in the vicinity of the matrix since the wickable material is pre-wetted with said alcohol.

Furthermore, according to certain embodiments, said alcohol is selected from the group consisting of: ethanol, pentanol, butanol, and nonanol.

Further, according to some embodiments, said sample is incubated in the presence of a plurality of concentrations of alcohol. According to another embodiment, the sample is incubated in the presence of a plurality of different types of alcohol.

Still further, according to a preferred embodiment, a chemical inhibitor of an aldehyde dehydrogenase enzyme is added during the incubation step. In such case, according to a preferred embodiment, said chemical inhibitor is cyanamide.

Additionally, according to a preferred embodiment, said sample is selected from the group consisting of: a sputum sample, a throat swab, a gastrointestinal sample, and a fermentation sample.

The present invention additionally provides a kit for measuring the ability of a sample, to metabolize alcohol, comprising: alcohol solution; blood cells or a blood substitute; an immobilizing solid support for supporting said blood cells.

According to a preferred embodiment of this kit, the blood cells or blood substitute are affixed to a paper disk solid support.

Further, according to a preferred embodiment of this kit, the blood cells or blood substitute are present within a matrix.

In a preferred embodiment, the kit further comprising calibration means for measuring and assessing the degree of alcohol metabolism of said sample. Preferably the calibration means are selected from a graph, a numerical table and a color scale.

The present invention additionally provides a method for screening a human for the risk of developing cancer, comprising: collecting a biological sample from said human, applying said sample into, upon, or adjacent to media comprising immobilized red blood cells, adding alcohol above, onto or adjacent to said media, incubating said media to allow growth of microorganisms, quantifying the degree of hemolysis occurring in said media, comparing the quantification results with a previously obtained standard, and evaluating the risk of said human for developing cancer. A large degree of hemolysis corresponds to a high risk for the development of said cancer.

Further, according to a preferred embodiment, said cancer is oral or pharyngeal cancer, and said sample is collected from the mouth or throat. In another embodiment, said cancer is in the gastrointestinal tract.

In a preferred embodiment of the screening method, the matrix is a selected from the group consisting of: agar, gelatin, cellulose, methylcellulose, nylon, paper and glass wool The present invention additionally provides a method for evaluating the cytotoxicity of an entity, comprising bringing an entity into contact with, or adjacent to, immobilized red blood cells or to an immobilized blood substitute, and ascertaining the degree of hemolysis of said red blood cells or blood substitute.

According to a preferred embodiment, said entity is selected from the group consisting of: a reagent, a biological material, and a cell producing an aldehyde.

Moreover, according to a preferred embodiment, said red blood cells are deficient in a predetermined enzyme.

Further, according to a preferred embodiment, the blood substitute is selected from the group consisting of: hemoglobin encapsulated by a membrane, hemoglobin encapsulated in a liposome, and a colored material encapsulated by a membrane.

There is also provided in the present invention, a method for evaluating the ability of matter to prevent cytotoxicity, comprising: bringing a cytotoxic agent into contact with immobilized red blood cells or with an immobilized blood substitute; adding said matter; measuring the degree of hemolysis; wherein a large degree of hemolysis is equated with the matter having low cytotoxicity prevention ability, and a small degree of hemolysis is equated with high cytotoxicity prevention ability.

According to a preferred embodiment of this method, the matter is selected from the group consisting of: a reagent, a biological material, a biological cell having aldehyde degrading capability, bacteria, yeast, fungi and plant cells.

Moreover, according to a preferred embodiment, the degree of hemolysis is evaluated in comparison to at least one control.

Further, in certain embodiments, the cytotoxic agent is volatile, and vapors of said cytotoxic agent are contacted with the immobilized red blood cells or blood substitute.

Still further, in certain embodiments, said red blood cells are deficient in a particular enzyme.

Additionally, in certain embodiments the blood substitute is selected from the group consisting of: hemoglobin encapsulated by a membrane, hemoglobin encapsulated in a liposome, and a colored material encapsulated by a membrane.

Moreover, according to a preferred embodiment, the matrix is selected from the group consisting of: agar, gelatin, cellulose, methylcellulose, nylon, paper and glass wool.

Additionally, according to a preferred embodiment, the cytotoxic agent is selected from the group consisting of an aldehyde, a peroxide and a ketone.

Further, in certain embodiments, a plurality of types of living cells are used, most of which do not posses cytotoxicity prevention abilities.

The invention further provides a method for evaluating the ability of a compound to lower the risk of a subject for developing cancer, comprising: collecting a biological sample from said subject; using the collected biological sample as the biological sample as above; incubating the matrix in the presence of said compound, quantifying the degree of hemolysis occurring in said matrix, comparing the quantification results with a previously obtained standard, and evaluating the ability of a compound to lower the risk of a subject for developing cancer, wherein a lowered degree of hemolysis is associated the ability of a compound to lower the risk of a subject for developing cancer. BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is herein described, by way of example only, with reference to the accompanying drawings, wherein:

Figure 1 shows hemolysis in a blood agar plate, around colonies of a highly hemolytic strain (Candida krusei). In addition, Figure 1 shows a protective strain (Candida albicans 904) preventing the hemolysis.

DETAILED DESCRIPTION OF THE INVENTION

It is appreciated that the detailed description that follows is intended only to illustrate certain preferred embodiments of the present invention. It is in no way intended to limit the scope of the invention, as set out in the claims.

A typical experiment using the method for identifying and measuring the ability of microorganisms within a sample, to metabolize alcohol, is now described. A blood agar plate (Columbia blood agar or tryptic soy blood agar, both obtained from Hy Labs, Rehovot, Israel) is inoculated in duplicate, either by streaking inoculation or by application of liquid samples of the microorganisms to be examined. Alcohol is added to one of each member of the pair of agar plates, by wetting absorbent paper (Whatman) attached to the inner side of the lid of the agar plate, with absolute ethanol. As a negative control, the microorganisms are grown at the same conditions, but in the absence of added alcohol. The agar plates are sealed with Parafιlm^Tm (Dupont) and incubated at various temperatures. In a variation of this experiment, the cells may be grown for a given time in the absence of alcohol vapors, and subsequently incubated in the presence of the alcohol. After 24 hours growth, an additional amount of alcohol may be applied to the absorbent paper. During or following growth, the hemolysis is observed in the immediate vicinity of the colonies grown in the presence of alcohol, and can be compared with the appearance of the duplicate plate grown in the absence of alcohol.

EXAMPLE 1

To test various microorganisms for their ability to cause hemolysis in the presence of ethanol, experiments were performed according to the above protocol, wherein 0.4 ml ethanol was initially applied to the filter set in the lid of the Petri dish. The cells were inoculated by streaking inoculation and incubation at 30÷ or 37÷ Celsius (for yeast and bacteria, respectively) under aerobic conditions, commenced concomitant with addition of alcohol. After 24 hours, another 0.2ml of ethanol was applied to the paper within the lid.

Microorganisms, which demonstrated ethanol-mediated hemolysis, included strains of:

Saccharomyces cerevisiae,

Candida albicans

Candida krusei

Gemella sp.

Helicobacter pylori

Microorganisms that were negative or provided a weak response in the presence of alcohol include strains of: Streptococcus faecalis Streptococcus salivarius

In the control lacking ethanol, hemolysis was not observed, or was very weak.

Among isolates obtained from air and soil, we have also observed this phenomon, in certain unidentified isolates.

Hemolysis of erythrocytes often stems from oxidative stress, or very low pH. The hemolysis observed here may be a direct or indirect consequence of enzymatic conversion of alcohol to its appropriate aldehyde, catalyzed by alcohol dehydrogenase present within the target microorganisms. Acetaldehyde is a strong oxidizing and reactive agent, and has been shown to lyse erythrocytes in liquid suspension. Aldehydes derived from alcohols can also act as inducers for other pathways that may be involved in this phenomenon. Alternatively, alcohol may be converted to other oxidizing agents. Several pieces of evidence suggest that the hemolysis observed here is related to oxidation. Firstly, we have found that this phenomenon is more pronounced on older, rather than fresher, blood agar plates. In fact, the phenomenon was initially discovered by using blood agar plates that were outdated (two months beyond the last date of use suggested by the manufacturers). Secondly, incubation of the blood agar plates for 24 hours in the presence of hydrogen peroxide vapor (applied as 100 microliter of a 30% solution onto filter paper attached to the lid of the petri dish) increased both the rate and extent of alcohol-mediated hemolysis. Thirdly, experiments with resazurin, a redox and pH indicator, showed that in the presence of ethanol and yeast, a rapid change to oxidative conditions was noted, as opposed to a very slow change to oxidative conditions in the presence of yeast with no alcohol added. This rapid change was mitigated in the presence of increasing concentrated of 4-methyl pyrazole, a known inhibitor of alcohol dehydrogenase. Similar results were obtained when alcohol dehydrogenase was employed rather than yeast cells. Finally, we found that hemoglobin itself was converted largely to its oxidized form (methemoglobin) in the presence of yeast cells and ethanol, as opposed to ethanol alone.

According to the hypothesis presented above, alcohol is first oxidized to the corresponding aldehyde, catalyzed by alcohol dehydrogenases. Such aldehydes may have a cytotoxic effect, hence the hemolysis. However, the aldehyde may be further oxidized (by aldehyde dehydrogenases), to yield the corresponding acids (e.g. acetic acid in the case of ethanol), in which case the hemolysis will not be as pronounced, or may not even occur. Both these reactions can be followed in vitro by spectrophotometric measurement ofNADH.

Alternatively, other mechanisms may be involved in the observed phenomenon. Aerobic degradation of alcohols is related to competent mitochondrial function, DNA repair mechanisms, iron uptake and metabolism, oxidation and reduction processes, stress, oxygen detoxification, membrane integrity, and cell-cell interactions. EXAMPLE 2

When a variety of yeast strains were observed, they differed radically in their hemolytic ability when plated on blood agar plates in the presence of ethanol. Some were highly hemolytic, others were not hemolytic at all; some became hemolytic only when cyanamide, an inhibitor of aldehyde dehydrogenase, was added. One implication is that when the second reaction (i.e., oxidation of aldehyde to acid is impeded, larger quantities of acetaldehyde build up in the vicinity of the microorganisms on the plate. The table below summarizes the hemolysis of various microbial strains in the presence or absence of ethanol and of cyanamide. The experiment was performed as described above. Cyanamide addition, where relevant, was performed as follows: after 24 hours of incubation, 20 microliters of a 1% aqueous solution of cyanamide (w/v) was applied over each streak. Addition of cyanimide in the absence of alcohol had no effect. Results were scored as follows:

0=no hemolysis l=slight greening (alpha) hemolysis below the microbial growth 2=greening hemolysis extending beyond the microbial growth 3=greening and clear (beta) hemolysis beyond the microbial growth

EXAMPLE 3

Several in vitro experiments were then performed to test whether there is indeed a correlation between hemolysis as seen in the petri dishes and between the generation of acetaldehyde in vitro. The generation of acetaldehyde was in essence observed by measuring the level of NADH, which is a second product of the reaction. Strains were grown in YPD (yeast extract/peptone/dextrose) medium (Difco) with either 0% or 5% ethanol v/v at 30° for 24 hours. Following growth, cells were centrifuged twice and resuspended each time in phosphate-buffered saline (PBS). Cells were suspended and samples added (250 microliters) to microtitre plate wells (96 wells, NUNC, Roskilde, Denmark) and adjusted to yield an optical density of approximately 1 OD (650 nm) as determined in a microtitre plate reader. Then cell suspension (25 microlitres) were mixed with NAD+ (final concentration 10 mM), glycine buffer (containing trapping agent, obtained from Sigma , St. Louis) (final concentration of glycine 0.1 M), ethanol (final concentration 40 mM) and water to a final volume of 250 microliters per well. The trapping agent is a chemical scavenger that does not allow the acetaldehyde to accumulate. Accumulation of acetaldehyde would inhibit the aldehyde dehydrogenase enzyme from producing additional acetaldehyde. Readings of the optical density were performed at 340 nm (suited to NADH). Results are shown for the 5000- second time point.

Optical density readings for Candida albicans strains 900, 904, 935, 952, 962 and Candida krusei. OD (difference from the value at time zero), after 5000 sec, at a wavelength of 340nm. Values were adjusted to correct for differences in the initial OD of the suspensions by dividing ΔOD at 5000 sec. by the initial OD. Hemolysis scores, graded according to the scale delineated above, are shown for comparison.

As seen in the table above, when the above six strains were compared, correlations were observed in most cases, between the relative level of acetaldehyde in the spectrophotometric assay and the degree of hemolysis. However, at least one glaring discrepancy was observed. Strain 904 did not produce hemolysis on blood agar plates in the presence of alcohol, yet still produced significant levels of acetaldehyde. If the acetaldehyde was indeed the factor causing hemolysis, this result was not easily understood.

One explanation the inventors considered is that strain 904 is highly protective, i.e., while it produces some alcohol dehydrogenase, it produces high quantities of aldehyde dehydrogenase, so that any acetaldehyde it produces is efficiently scavenged, thus no hemolysis occurs in the plates.

If this was indeed the case, then strains putatively producing high amounts of aldehyde dehydrogenase should 'protect' blood agar from hemolysis in the presence of hemolytic strains. To test this hypothesis, colonies of highly hemolytic strains (Candida krusei and Candida albicans 962) were plated next to two putative protective strains (Candida albicans 904 and 950) on the blood agar. Indeed, the protective strains prevented hemolysis of the hemolytic strain in its vicinity (refer to Figure 1, described below).

Several lines of evidence support the conjecture that acetaldehyde is directly or indirectly involved in the hemolysis of the blood agar. Firstly, addition of acetaldehyde alone to the blood agar plates (with no microorganisms present) yields a color change reminiscent of alpha-hemolysis. Secondly, addition of cyanamide to the blood agar greatly increased the rate and extent of hemolysis in many cases, presumably by inhibiting the enzyme (acetaldehyde dehydrogenase) that normally catalyzes the conversion of acetaldehyde to acetate.

Furthermore, addition of NaOH and zinc ions individually and simultaneously, both of which increase alcohol dehydrogenase activity, increased the extent of hemolysis in the presence of C. krusei. When yeast cells were inoculated onto 0.45 micron (pore size) nitrocellulose filters placed on blood agar plates in the presence of ethanol, hemolysis was observed beneath the filters. This demonstrates that the agent causing the hemolysis can diffuse through a filter to the plates. This provides additional, albeit indirect support for the above proposal.

In earlier studies, the inventors found that certain alcohols spontaneously cause hemolysis on the blood agar plates, without the presence of any microorganisms. One of these was n-nonanol (C9 alcohol). When vapors of nonanol were added to a plate that contained colonies of strain 904, the Candida albicans 904 protected the red blood cells from hemolysis, in the vicinity of the colony. In other words, the strain protected the blood cells from lysis by the n-nonanol. The hypothesis was proposed that nonanol acts to lyse red blood cells because it is oxidized to the C9 aldehyde, (perhaps by enzymes present in the erythrocytes). The hypothesis further suggests that aldehyde dehydrogenase from the protective strain diffuses from the colony and protects the surrounding blood cells from hemolysis. This hypothesis was supported, as follows. Cyanamide was added to blood agar plates containing colonies of Candida albicans 904. The plates were incubated in the presence of n-nonanol vapors (20 microliters of n-nonanol added to a paper disc on the lid of the plate). The cyanamide reduced the protection, suggesting that inhibition of the aldehyde dehydrogenase destroyed the protective effect.

Referring to Figure 1, a highly hemolytic strain (Candida krusei) was streaked in the center (B) of a blood agar plate. At either end, two streaks of a protective strain (Candida albicans 904) were made (C and D). 20 microlitres of 1% cyanamide solution was added to the streak on the left (C). As expected, a halo of hemolysis was observed following two days incubation surrounding the Candida krusei growth (E). Streak (C) of C. albicans 904 prevented hemolysis of the hemolytic strain in its vicinity, but the streak on the left (D), to which cyanamide was added, did not protect the red blood cells from hemolysis.

A =unaffected blood cells

B =Candida krusei

C = Candida albicans 904 D = Candida albicans 904 with 20 microlitres cyanamide 1% E =hemolysis halo

Based on these results, the inventors hereby disclose a general method for evaluating if an entity is cytotoxic or not, and whether this toxicity can be reversed. According to this method, an entity is deemed harmful to biological cells or to animals if it brings about lysis of immobilized red blood cells. In this method, the entity undergoing evaluation is added to red blood cells that have been immobilized within a matrix. After a predetermined incubation period, the degree of hemolysis of said the blood cells is measured. In a preferred embodiment, the degree of hemolysis is then compared to a known standard. A large degree of hemolysis is equivalent to high cytotoxicity, and a low degree of hemolysis is equivalent to minimal cytotoxicity. This method is highly versatile, and is useful to examine the cytotoxicity of widespread materials; anything from an unknown plant cell to radiation or to chemical compounds can be examined, for example, using this method.

One preferred matrix, within which the red blood cells are immobilized, is agar. A standard blood agar microbiology media can be used, and standard blood agar plates can be purchased for this purpose. Alternatively, if the entity undergoing evaluation has an uneven or a large surface, liquefied blood agar can be poured or applied over the surface of the entity, to gel upon this surface. The entire entity can then be incubated to see if hemolysis occurs. Areas of both (-hemolysis and (-hemolysis represent cytotoxicity. Other types of matrices are known within which red blood cells can be immobilized. These include, but are not limited to, gelatin, cellulose, methylcellulose, nylon, paper and glass wool.

In certain embodiments, a blood substitute is used instead of red blood cells. This blood substitute can be any colored substance that is contained within vesicles that can then be lysed by toxic materials. Examples include hemoglobin encapsulated by a membrane, or within a liposome, or a dye encapsulated by a membrane. In one embodiment, red blood cells are used which are deficient, or differ, in specific enzymatic pathways, making these blood cells more susceptible, or less susceptible to hemolysis. It is clear that red blood cells from a wide variety of mammalian origins can be used, having diverse isoenzymes, and different metabolic pathways. Though the inventors have utilized sheep red blood cells, the invention is not limited to red blood cells of this origin.

In certain embodiments, the entity undergoing evaluation is placed adjacent to the matrix, with no direct contact between the entity and the matrix. This would allow vapors of a volatile entity to reach the matrix, and undergo evaluation. An example of volatile entity would be an aldehyde, such as acetaldehyde.

The invention additionally discloses a general method for evaluating the ability of matter to prevent cytotoxicity. According to this method, a cytotoxic agent is brought in contact with immobilized blood cells. A substance undergoing evaluation is added, to see if its addition can protect the red blood cells from being lysed. If no lysis occurs, or if the degree of lysis is lessened (as compared with appropriate controls), the substance (or matter) undergoing evaluation is deemed to have cytotoxicity prevention ability. A small degree of hemolysis is equated with high cytotoxicity prevention ability, and a large degree of hemolysis is equated with the matter having low cytotoxicity prevention ability.

The present invention discloses a method for identifying and measuring the ability of microorganisms within a sample, to metabolize alcohol, based on the degree of hemolysis observed in red blood cells immobilized within a matrix. The sample is added to the matrix, alcohol or alcohol vapors are added in the vicinity, and the matrix is incubated for a specific amount of time. The degree of hemolysis is measured; a large degree of hemolysis is equated with high alcohol metabolism abilities, and a small degree of hemolysis is equated with low alcohol metabolism abilities.

The ability to rapidly detect microorganisms metabolizing alcohol is of considerable use in the diagnostic detection of cancer risk among alcohol drinkers, mouthwash users, and the general public. In such an instance, a sample, such as saliva, would be incubated in the presence of blood, for instance on agar plates, and the degree of hemolysis assessed. In the simplest scenario, this would take place on a blood agar plate, however different matrices or solid supports may be considered. For example, the present invention provides a kit, in which for instance, a saliva sample can be mixed with a drop of blood and applied, for instance, to a paper disk. Hemolysis is determined by the diffusion of hemoglobin away from the site of application.

In addition to the rapid detection of putative cancer-related microorganisms, the methods of the present invention have potential application in the area of fermentation technology. One of the major problems in alcohol fermentation is finding strains capable of withstanding high ethanol concentrations. One of the key enzymes in this process is the same enzyme that converts the ethanol in the cells to acetaldehyde. Thus, we anticipate that clones differing in alcohol dehydrogenase will be readily identifiable by production of different sizes or kinds of halos of hemolytic activity. Similarly, cells producing different quantities or kinds of aldehyde dehydrogenase may be important in preventing the toxic effects of acetaldehyde. These cells can be identified, for example, by their ability to protect against hemolytic strains, or to prevent hemolysis in the presence of n- nonanol or other alcohols.

Furthermore, in a more general context, this invention contains a simple way to test for microbial transformations of molecules into more toxic compounds. Application of a nontoxic compound to microorganisms on a blood agar plate can be followed by production of a toxic compound leading to hemolysis. Microorganisms can be screened in this manner for production of toxic by-products. Conversely the inverse of this technique can also be used to search for de-toxifying microorganisms, in which case instead of creating a hemolytic halo, the microorganisms (e.g. colonies) would create a halo of protection.

The following table summarizes the ability of certain microorganisms to 'protect' the blood agar from hemolysis in the presence of n-nonanol vapors.

In previous unpublished results, there were occasional colonies of microorganisms that 'protected' the blood agar from 'spontaneous' lysis over time. We now think that such strains may be high in aldehyde dehydrogenase, and thus protected the blood. If so, perhaps lysis of the blood cells was due to aldehyde generated within the erythrocytes themselves. This has many repercussions.

Claims

1. A method for determining metabolism of a given metabolite, comprising the steps of: a) adding a biological sample adjacent to or upon a matrix, said matrix comprising immobilized blood cells or a blood substitute; b) incubating said matrix in the presence of said metabolite; c) assessing the degree of hemolysis occurring in said matrix, wherein a large degree of hemolysis is equated with a high degree of metabolism of said metabolite, and a small degree of hemolysis is equated with a low degree of metabolism of said metabolite.

2. The method according to claim 1, wherein the metabolite is added in vapor form.

3. The method according to claim 1, for identifying and measuring the ability of a sample, to metabolize or modify alcohol, comprising steps (a), (b), (c) of claim 1, wherein in step (b), incubating the matrix in the presence of said metabolite, alcohol is the metabolite, which is added above, onto or adjacent to said matrix; and wherein a large degree of hemolysis is equated with high alcohol metabolism abilities, and a small degree of hemolysis is equated with low alcohol metabolism abilities.

4. The method according to claim 3, wherein the alcohol is added in vapor form.

5. The method according to claim 4, wherein said alcohol vapors originate from a wickable material placed in the vicinity of the matrix; said wickable material being pre- etted with said alcohol.

6. The method according to claim 3, wherein said alcohol is selected from the group consisting of: ethanol, pentanol, butanol, and nonanol.

7. The method according to claim 3, wherein said sample is incubated in the presence of a plurality of concentrations of alcohol.

8. The method according to claim 3, wherein said sample is incubated in the presence of a plurality of different types of alcohol.

9. The method according to claim 3, wherein a chemical inhibitor of an aldehyde dehydrogenase enzyme is added during the incubation step.

10. The method according to claim 9, wherein said chemical inhibitor is cyanamide.

11. The method according to claim 3, wherein said sample is selected from the group consisting of: a sputum sample, a throat swab, a gastrointestinal sample, and a fermentation sample.

12. The method according to claim 1, wherein said biological sample comprises microorganisms.

13. The method according to claim 1, wherein said matrix is a selected from the group consisting of: agar, gelatin, cellulose, methylcellulose, nylon, paper, glass wool, alginate, guar gum, xanthan and carboxymethylcellulose.

14. The method according to claim 1, wherein said blood cells are aged to sensitize said blood cells to hemolysis.

15. The method according to claim 1, wherein said matrix is a semi-solid.

16. A kit for measuring the ability of a sample, to metabolize alcohol, comprising: a) alcohol solution; b) blood cells or a blood substitute; c) an immobilizing solid support for supporting said blood cells.

17. The kit according to claim 16, wherein said blood cells or blood substitute are affixed to a paper disk solid support.

18. The kit according to claim 16, wherein said blood cells or blood substitute are present within a matrix.

19. The kit according to claim 16, further comprising calibration means for measuring and assessing the degree of alcohol metabolism of said sample.

20. The kit according to claim 19, wherein said calibration means are selected from a graph, a numerical table and a color scale.

21. A method for determining whether a subject has a high risk of developing cancer, comprising: collecting a biological sample from said subject; using said collected biological sample as the sample of claim 3; quantifying the degree of hemolysis occurring in said matrix, comparing the quantification results with a previously obtained standard, and evaluating the risk of said human for developing cancer, wherein a large degree of hemolysis is associated with a high risk for the development of said cancer.

22. The method according to claim 21, wherein said cancer is oral or pharyngeal cancer, and said sample is collected from the mouth or throat.

23. The method according to claim 21, wherein said cancer is in the. gastrointestinal tract.

24. The method according to claim 21, wherein said matrix is a selected from the group consisting of: agar, gelatin, cellulose, methylcellulose, nylon, paper and glass wool.

25. A method for evaluating the cytotoxicity of an entity, comprising bringing an entity into contact with, or adjacent to, immobilized red blood cells or to an immobilized blood substitute, and ascertaining the degree of hemolysis of said red blood cells or blood substitute.

26. The method according to claim 25, wherein said entity is selected from the group consisting of: a reagent, a biological material, and a cell producing an aldehyde.

27. The method according to claim 25, wherein said red blood cells are deficient in a predetermined enzyme.

28. The method according to claim 25, wherein said blood substitute is selected from the group consisting of: hemoglobin encapsulated by a membrane, hemoglobin encapsulated in a liposome, and a colored material encapsulated by a membrane.

29. A method for evaluating the ability of matter to prevent cytotoxicity, comprising: a) bringing a cytotoxic agent into contact with immobilized red blood cells or with an immobilized blood substitute; b) adding said matter; c) measuring the degree of hemolysis; wherein a large degree of hemolysis is equated with the matter having low cytotoxicity prevention ability, and a small degree of hemolysis is equated with high cytotoxicity prevention ability.

30. The method according to claim 29, wherein said matter is selected from the group consisting of: a reagent, a biological material, a biological cell having aldehyde degrading capability, bacteria, yeast, fungi and plant cells.

31. The method according to claim 29, wherein the degree of hemolysis is evaluated in comparison to at least one control.

32. The method according to claim 29, wherein said cytotoxic agent is volatile, and vapors of said cytotoxic agent are contacted with the immobilized red blood cells or blood substitute.

33. The method according to claim 29, wherein said red blood cells are deficient in a particular enzyme.

34. A method according to claim 29, wherein said blood substitute is selected from the group consisting of: hemoglobin encapsulated by a membrane, hemoglobin encapsulated in a liposome, and a colored material encapsulated by a membrane.

35. The method according to claim 29, wherein said matrix is selected from the group consisting of: agar, gelatin, cellulose, methylcellulose, nylon, paper and glass wool.

36. The method according to claim 29, wherein said cytotoxic agent is selected from the group consisting of an aldehyde, a peroxide and a ketone.

37. The method according to claim 29, wherein a plurality of types of living cells are used, most of which do not exhibit cytotoxicity prevention abilities.

38. A method for evaluating the ability of a compound to lower the risk of a subject for developing cancer, comprising: collecting a biological sample from said subject; using said collected biological sample as the biological sample of claim 1 ; incubating the matrix in the presence of said compound, quantifying the degree of hemolysis occurring in said matrix, comparing the quantification results with a previously obtained standard, and evaluating the ability of a compound to lower the risk of a subject for developing cancer, wherein a lowered degree of hemolysis is associated the ability of a compound to lower the risk of a subject for developing cancer.