Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/02—Investigating particle size or size distribution
  • G01N15/0205—Investigating particle size or size distribution by optical means
  • G01N15/0227—Investigating particle size or size distribution by optical means using imaging; using holography
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/02—Investigating particle size or size distribution
  • G01N15/0205—Investigating particle size or size distribution by optical means
  • G01N15/0211—Investigating a scatter or diffraction pattern
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/47—Scattering, i.e. diffuse reflection
  • G01N2021/4704—Angular selective
  • G01N2021/4711—Multiangle measurement

Definitions

• the present invention is directed to the field of bioassays, and in particular to an assay utilizing radiation, or laser, technology in combination with unique genetically engineered bacteria that provide a "fingerprinting" capacity to identify and measure concentrations of toxicants in various environmental media.
• sample preparation can be complex and different assays must be developed for the identification and quantification of individual chemicals or chemical classes.
• knowledge that a chemical is present and its concentration does not indicate its cytotoxic or genotoxic activity.
• the most widely employed bacterial test system for carcinogens is the Ames test, Ames, Mutat. Res. 31:347- 349 (1974), using Salmonella typhimurium histidine- requiring auxotrophs. Because the Ames assay relies on reverse mutation, a mutant deoxyribonucleic acid (DNA) locus must be mutated back to its wild-type configuration or be suppressed. Thus, chemical mutagens that cannot effect this change in the DNA will go undetected.
• researchers have recognized other shortcomings in this test, specifically an inability to devise an in vitro activation system that is reliable and reproducible, and an inability to detect some mutational events that can lead to cancer. Felkner, Microbial Testers; Probing Chemical Carcinogenesis, 177 (1981).
• Felkner discovered that ordinary transforming strains of Bacillus subtilis recovered from short exposures to the potent mutagenic and carcinogenic agent, 4-nitroquinoline-l-oxide (4NQO), while other strains incapable of recombination and/or dark repair did not.
• the ability or inability to recover was found to be directly correlated with the blocking of de novo deoxyribonucleic (DNA) synthesis through complexation of 4NQ0 with the DNA of the cell.
• DNA de novo deoxyribonucleic
• the product of this research was the discovery that a certain chemical structure has a corresponding particular biological function. Based on this principle, the effect of a chemical on a biological system, such as Bacillus subtilis bacteria, may be identified by the change in morphology of the assayed cells.
• Felkner developed a microbial bioassay from a collection of several hundred Bacillus subtilis mutants based on the principles derived from his earlier work. These selected strains had unique genetic defects causing them to be very sensitive to the effects of specific chemical classes, toxins and radiation. To enable uniformity, the mutant genes from these strains were introduced by recombination technology into identical clones of the wild type (normal) Bacillis subtilis strain. Thus, a battery of strains, differing only by a single character (gene) or limited set of genes was produced. The member strains are therefore isogenic, that is, identical except for single defined genes.
• Bioassays employing bacteria are disclosed in Wyatt U.S. Patent No. 3,730,842, issued May 1, 1973, and Wyatt U.S. Patent No. 4,101,383, issued July 18, 1978. These patents disclose methods of determining bacterial sensitivity or susceptibility to antibiotics by using test differential scattering patterns derived from the test bacteria and then comparing them to patterns of control bacteria. Neither reference provides for generating a pattern to screen for a toxicant having potential cytotoxic or genotoxic activity or to provide a means by which the toxicant may be identified and quantified.
• Wyatt U.S. patent No. 4,541,719, issued September 17, 1985 discloses a laser apparatus that measures scattered radiation by microparticles such as bacteria at two well-defined scattering angles.
• Another object of this invention is to provide unique differential light scattering (DLS) patterns for chemical agents using isogenic repair mutant and wild type strains of Bacillus subtilis.
• DLS differential light scattering
• a further object of this invention is to select from the Bacillus subtilis strains a minimum group or "isoset" required to establish response patterns to specific chemical classes.
• This invention has as yet another object the provision of a means to score the toxicity responses of the selected bacteria and to compare these responses to the responses of these bacteria to known chemical agents.
• Another object of this invention is to establish means for evaluating toxicity responses for compounds whose genotoxic responses have been difficult or even impossible to detect with other state-of-the-art mutagenicity assays.
• This invention has the further object to provide, as a reference, DLS patterns generated when these bacteria are treated with identified chemicals, to screen for toxicity and to identify and quantify samples.
• a further object of this invention is the provision of a medium for solubilizing water insoluble samples to be tested, this medium being harmless to the assay bacteria and being optically clear when mixed with the bacteria so that radiation may readily penetrate, and scattering may be detected.
• the foregoing and other objects of the invention are achieved by the provision of Bacillus subtilis isogenic strains of bacteria lacking certain genetic repair functions and strains that are repair efficient that can be monitored for their unique responses to toxic chemicals using a differential light-scattering laser system. These responses are then scored to determine, for example, acute toxic or genotoxic effects. These scores, calculated as response indices, are then compared to known responses of these bacteria strains to identified chemical agents. It is to be understood that this method is also capable of assessing quantifiably the presence of a chemical to be tested.
• Figure 1 is a schematic view of the method and apparatus of the claimed invention.
• Figures 2a through 2d are differential light scattering patterns generated when the assay bacteria is exposed to certain concentrations of 4-nitroquinoline-N-oxide (“4NQO").
• Figures 3a and 3b are differential light scattering patterns generated when the assay bacteria is exposed to certain concentrations of N-methyl-N'-nitro-N-nitroso-quanadine ("MNNG").
• MNNG N-methyl-N'-nitro-N-nitroso-quanadine
• Figures 4a through 4d are differential light scattering patterns generated when the assay bacteria is exposed to certain concentrations of benzo(a)pyrene ("B(a)P").
• Figures 5a through 5d are differential light scattering patterns generated when the assay bacteria is exposed to certain concentrations of "Lindane”.
• FIG. 1 provides a schematic illustration of the claimed apparatus by which samples are bioassayed in accord with the claimed invention.
• the bioassay consists of a radiation source 4, such as a laser system, which in the preferred embodiment employs the laser system such as that disclosed in Wyatt U.S. Patent No. 4,541,719, which includes a laser producing monochromatic visible or infrared radiation that is plane polarized with respect to two arrays of detectors.
• the laser system is capable of making 1200 measurements/second at 15 unique angles.
• the bioassay further includes an isogenic set of Bacillus subtilis mutants 2. The laser illuminates the cuvette 1 containing the bacteria 2 in suspension, these bacteria causing the light to be differentially scattered.
• the differential light scattering (DLS) 6 of the laser beam is detected as scattered light intensity as a function of angle.
• the detectors 5 detect the DLS and feed the signals to a recorder 7, which records, in the form of a graph, a plot of the scattered intensity.
• an analysis apparatus such as a computer 8 receive the data from the detectors 5 directly.
• the intensity variation in the scattered light is called the DLS pattern, and is dependent on the average size and structure of the bacteria as well as the population size distribution.
• the DLS pattern of a suspension shows how many particles are present, their size, shape and the distribution of particles.
• Bacterial count is expressed as a measure of height, or the highest point, of the DLS pattern.
• the shift of peaks of a sample DLS pattern indicates the degree of morphological change as compared to the control pattern.
• the DLS pattern may be in magnetically recorded digital form for machine readability, or recorded in graphic form such that the scattered intensity pattern is represented as an eye readable curve.
• a digitizing tablet (not shown) is used to trace each pattern's curve and simultaneously transmit a continuous stream of x and y coordinates to an analysis apparatus such as a computer 8.
• an analysis apparatus such as a computer 8.
• the analysis apparatus which is in the preferred embodiment a computer 8 carries out the required calculations of response indices representing the shift in the DLS of the control as compared to the sample, and the change in bacterial count.
• the derived indices provide an immediate preliminary indication of toxic response, the DNA damaging (genotoxic) potential and aid in the identification of "isosets" of the Bacillus subtilis bacteria for compound identification.
• the concentration of the chemical sample is determined by the level of response by the bacteria to the given sample.
• the set of bacteria selected for the bioassay each differ by only one property, which causes them to be more sensitive than other set members because of the mechanism of toxicity. Therefore, a "fingerprint", or profile unique to each chemical is generated from the differential response of the member bacteria. By "scoring" these fingerprints, the bioassay provides data for identifying test chemicals, assessing the concentration of test chemicals, and assessing whether the test chemical is acutely or chronically toxic.
• Another embodiment of the invention provides a library 9 wherein are stored the DLS patterns and calculated response indices for the bioassay of identified samples. By comparing the sample DLS with the stored pattern, the sample may be screened for toxicity, identified, and/or quantified.
• the time required for a complete assay is approximately sixty (60) to sixty-six (66) minutes, the time required for untreated bacteria to divide twice in the absence of any chemical sample. However, individual samples may be analyzed at a rate of one sample per minute to determine the concentrations of a chemical in an aqueous sample.
• the Bacteria The bacteria used in the bioassay are of a single species. Bacillus subtilis, which has numerous advantages over other bacteria traditionally used in bioassays. First, Bacillus subtilis is the most widely studied and genetically mapped gram-positive microorganism. Henner, D.J. and Hoch, J.A., The Bacillus subtilis Chromosome, Microbiol. Rev. 44:57-82 (1980).
• Bacillus subtilis exists as a spore that is capable of surviving genetically unchanged while resisting heat, drying or other adverse effects for an indefinite period. As a vegetative cell, the bacteria can divide rapidly to double itself in approximately twenty-six (26) minutes during the logarithmic growth phase, and is much more sensitive at this stage to chemical toxicants than are the gramnegative microorganisms. Salmonella typhimurium and Escherichla coli. In its competent state, Bacillus subtilis readily takes up large molecules such as DNA, and can integrate genes derived from members of the same species or other species through traditional recombinant DNA technology.
• the bioassay uses Bacillus subtilis mutants that are genetically identical, or isogenic, except for one or more genetic blocks in unique enzymatic repair processes or steps that restore chemically damaged DNA to its functional condition.
• Bacillus subtilis are specifically selected strains deficient in different recombination (Rec-), excision (exc-), polymerase (Pol-) or spore repair (Spp-) repair steps.
• These strains may be deficient in one or a multiple of repair steps, and include the Rec- strains, recAl, recA8, recB2, recC5, recD3, recE4, recG13, mc-1, and m45; the Pol- strains T-1, TKJ8201; the Excstrains her-9 and TKJ8206; The Exc- and Rec- strain FH2006-7; the Exc-, Rec- and Pol- strain HJ15; the Exc- and Spp- strain TKJ5211; the Exc-, Pol- and Spp- strain TKJ6321; and repair efficient strains HA101, 168 and 168 wild type.
• the mutants in this group are therefore more sensitive to the metabolic toxicity or genotoxic activity or a broad range of chemical substances at nannomolar or nannogram concentrations.
• Bacillus subtilis mutants were developed by treating them with by irradiation or with various chemical mutagens. For example, nitrosoquanidine, a powerful mutagenic chemical, was used to change the genetic makeup of the bacteria and trimethoprim to select for mutant types that could not synthesize DNA unless thymine was provided to them.
• the mutants from one group isolated by Felkner were designated FH2001 through FH2006 and had been derived from a parent strain JBO1-200 whose lineage is defined in Felkner J. Bacteriol. 104:1030-1032 (1970).
• trp+, thy+, her-, rec- Types 2 and 3 are mutants suitable for assaying her- and rec- mutations to detect DNA damage. These bacteria, constructed by conventional genetic engineering techniques would not occur in nature or result in a surviving bacterial isolate.
• recA8, recA1, recB2, recC5, recD3, recE4, recG13, hcr-9, mc-1 and FH2006-7 are descended from strain 168. This strain is a descendent from the strain trp C of John Spizizen. Spizizen, J. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate, Proc. Nat. Acad. Sci.
• Isosets defined as a subset of these mutants, constitute the minimum number required to define a response to a sample tested.
• Data derived form fractional survival tests and data derived from spot assays are used to determine which B. subtilis strains should be selected to comprise an isoset to identify a given compound by the radiation bioassay.
• the isogenic mutants are pre-screened using the spot test with given chemical samples.
• the DNA-damaging test is performed according to the procedure disclosed in Felkner, I.C., Microbial Testers: Probing Carcinogenesis (1981) and Felkner, I.C. in Medine, A., and Anderson, W. , eds. Proceedings of the National Conference on Environmental Engineering, Am. Soc. Civil Eng., N.Y. pp. 204-209 (1983).
• Each mutant is inoculated into 5 ml of Brain Heart Infusion (BHI) broth, either from a disc containing approximately 1 ⁇ 10 7 washed spores or a 1 mm loopful from a sporulating slant culture, and incubated by shaking at 37 to 39° C for 16 hours.
• This culture is designated o/n.
• inocula from the o/n cultures are streaked radially on a nutrient agar plate to a sensitivity disc containing 10 ul of the assay chemical which may or may not have been pre-incubated with a rat liver microsomal fraction (S-9) in order to achieve metabolic activation.
• Liver microsomes designated an S-9 fraction, were prepared from Sprague-Dawley rats induced with Aroclor 1254 (Litton Bionetics, Washington, SC 19405). The S-9 fraction was prepared as a KCl homogenate with a protein concentration of 25-28 mg/ml. It is understood that this fraction may be microsomal or post-microsomal and is not limited by the origin of the tissues.
• a "cocktail” was therefore derived so that a clear aqueous solution of the test chemical could be mixed with the assay bacteria and the hydrophilic S-9 fraction, thereby minimizing interference with the radiation source and its ability to generate a DLS pattern of the radiation scattered by said bacteria.
• the "cocktail” has the advantage of solubilizing chemicals in an aqueous system that were not as readily solubilized by other known procedures using dimethylsulfoxide (DMSO), ethanol or acetone before being introduced into the aqueous assay mixture.
• DMSO dimethylsulfoxide
• acetone acetone
• the preferred embodiment of the bioassay employs a dispersant sold under the trademark "COREXIT 7664 Oil Dispersant” (Exxon Chemicals, Clark, New Jersey) and a non-ionic surfactant sold under the trademark “EMULPHOR EL-620” (GAF Corp., 140 West 51 Street, New York, N.Y. 10020). Both the "COREXIT” dispersant and the “EMULPHOR” surfactant are readily obtainable at the present time, and are generally known to chemists and commercial users. The chemical compositions of each of these are maintained as trade secrets by the respective owner companies.
• the "EMULPHOR” surfactant is a polyoxylated vegetable oil.
• the radiation source 1 of the bioassay is a laser such as that disclosed in Wyatt, U.S. Patent No. 4,541,719. This instrument records scattering intensity at fifteen angular locations, permitting the relationships between refractive index, size and shape to be measured. Moreover the read head is anodized, sealed and attached directly to the laser head as well as to an integrated circuit board, providing a unitized optical bench and a grounded enclosure for the detector elements.
• Computer interface Parallel analog outputs interfaced to 16-channel, 12 bit A/D programmable multiplexer.
• Detectors Transimpedance photodiodes with built-in amplifiers Detector Output Dynamic Range: 0 - 10 V Dynamic Range: 10 4 Linearity: ⁇ 0.1%
• the chemical B(a)P is a well known procarcinogen/mutagen that is insoluble in an aqueous system, and requires metabolic activation by rat liver microsomal fractions (S-9) to become electrophilic and to cause DNA damage or a mutagenic response.
• "Lindane” is a chemical difficult to assay for its genotoxic potential by state of the art mutagenic assays.
• Lyphilized tablets containing the appropriate Bacillus subtilis strains and dehydrated growth medium are introduced into cuvettes containing water. Suspensions of bacterial cells at the required concentration described in the examples provided infra may also be used.
• the inoculated cuvettes are placed in an incubation chamber that has been prewarmed to approximately 37 to 39° C, and incubation continued for about one hour to one and a half hours to establish a culture in the "assay-ready condition".
• Test or control material is introduced into the cuvettes using one pair of cuvettes for each assay organism.
• a negative control having no test material, and a positive control having an identified toxic chemical at a concentration whose response has been determined is included.
• the cuvettes are incubated for six minutes at 37-39° C, and each cuvette is read a minimum of fifty
• the bacteria could be lyphilized vegetative cells or spores in a capsule form. However, when spores are used, this form being preferred because of their stability, they must be heat-shocked at 60 degrees
• Centigrade Include a negative control having no test material, and a positive control having a toxic chemical at a concentration whose response has been determined.
• a negative control may also comprise a control sample of bacteria exposed to 4-nitroquinoline-N-oxide or 4-nitroquinoline-1-oxide and a positive control of bacteria exposed to benzo(a)pyrene.
• Cultivation of bacterial cells appropriate in concentration for the bioassay includes heat shocking strains of Bacillus subtilis in 1 ml of Brain Heart Infusion (BHI) broth for 20 minutes. Additional broth is added until an absorbance of 0.2 A(540) is achieved. The cultures are incubated at 37 to 39° C until the bacterial growth reaches an A(540) of 0.5, a period of approximately 2 hours.
• Cultures that require a longer period to reach this density are not considered suitable for the bioassay. Cultures found suitable are diluted to an A(540) of 0.3 with BHI broth yielding approximately 2 ⁇ 10 7 bacteria/ml. These cells, when diluted by the amount required for the cuvette assay, yield a cell concentration of 10 6 bacteria/ml.
• 4NQO was assayed at final concentrations of 8.5, 4.2 and 2.1 ug/ml using Bacillus subtilis strains recE4 (6), 168 or 168 wild type (11).
• the DLS patterns for each of these assays comprise Figures 2a through 2d.
• MNNG was assayed at a final concentration of 3.6 ug/ml with strains 6 and 11.
• the DLS patterns for each of these assays comprise Figures 3a and b.
• a screw cap tube containing 13.6 ml of deionized water at 37 to 39° C is pipetted 0.3 ml of bacteria and 0.1 ml of the test chemical.
• the tube is inverted gently and its contents poured into a cuvette.
• the cuvette is illuminated by the laser and the intensities of scattered light measured and scored.
• the cuvette is then incubated at 37-39 degrees
• the method may also be performed using lyphilized bacteria having with it sufficient dehydrated BHI to support the growth during the assay period.
• the total volume of fluid required to obtain a reading from the cuvette is 10 ml. Therefore, a volume of about 0.4 ml of fluid will rehydrate the cell-medium.
• the incubation period remains unchanged, and illuminating is performed at fifteen minute intervals.
• B(a)P was assayed at final concentrations of 15.1 and 7.6 ug/ml using Bacillus subtilis strains 11 and TKJ6321 (19).
• B(a)P was assayed at final concentrations of 15.1 and 7.6 ug/ml using Bacillus subtilis strains 11 and TKJ6321 (19).
• the DLS patterns generated by this assay comprise Figures 4a-4d.
• 180 ul each of "EMULPHOR” and “COREXIT” are added to each 5 ml of deionized water and mixed.
• 180 ul of S-9 fraction is added along with appropriate amount of lyphilized bacteria and medium required to achieve a cell concentration of 10 6 bacteria/ml.
• the scoring techniques of the bioassay measure the height of the DLS pattern as a function of bacterial count and the amount of shift in the peaks of the chemical sample pattern and the control sample pattern to determine the degree of morphological change in size and shape of the bacteria.
• the derived indices provide an immediate preliminary indication of the toxic response, the DNA damaging (genotoxic) potential, and identify isosets for compound identification.
• each pattern's height provided an indication of bacterial count and is therefore the principal indicator of toxicity.
• scoring techniques for the identification of morphological changes it was noted that there is a function which is continuous on a closed interval from the beginning of the pattern to its end. Each line segment between any two adjacent points formed an arc, and associated with each point on the X-axis is only one point on the pattern. Using a digitizing tablet, it is possible to obtain a sufficiently large number of points so that the sum of the distances between all adjacent points would approximate the length of the pattern. This was defined as the arc length of the curve. Variances of the arc length correspond with the variances in the bacteria morphology, especially as they related to individual cell size.
• the percent change between the test pattern's arc length and the baseline pattern's arc length was calculated. To determine the fourth order arc index, the arc percent change was divided by 5 and, if the change was positive, 1 was added. If the change was negative, 2 was subtracted. The integer value was retained.
• Each of the three digits, from left to right in the PRI index, derives from the following table according to the fourth order indices of the patterns, starting with the control at "0" time as the reference and the control at 60 minutes as the first digit.
• the tertiary responses for each of the indexed patterns were obtained by multiplying the fourth order Y index by the fourth order arc index.
• the secondary response index (SRI) was determined by the calculation of the sum of the tertiary indices for all indexed patterns combined. This method of calculation is illustrated by the following data derived from an assay:
• the interpretation of the PRI of 132 is that the control of sixty-six (66) minutes displayed normal growth, hence the number 1 as the first digit.
• the treated sample at time "0" showed a loss in bacterial count accompanied by size increase, hence the number 3 as the second digit.
• sixty-six minutes the treated sample showed an increase in bacterial count accompanied by a size increase, hence the number 2 in the third digit.
• PRIs Two extreme examples of PRIs are 133 and 144. These examples are immediately toxic responses from which the cells fail to recover.
• the 133 PRI shows cell enlargement, and the 144 PRI shows cell shrinkage, both being associated with the failure of the cell to recover but by different mechanisms of action.
• the presence of a 3 or 4 in the first digit of any PRI should mean that the assay is invalid because the bacterial control culture is not displaying normal growth.
• a screening score S provides a precise measure of bacterial growth or inhibition of bacteria in the test sample as compared to the control bacteria. It is defined in terms of DLS placement scores and was implemented by previous studies. Wyatt, P.J., Phillips, E.T., Scher, M.G., Kahn, M.R., and Allen, E.H., J. Argic. Food Chem. 25:1080 (1977); Wyatt, P.J., Scher, M.G., and Phillips, D.T., J. Agric. Food Chem. 25:1086 (1977). Thus,
• Dbc is the DLS displacement of the assay broth plus bacteria relative to the assay broth (control)
• Dc is the displacement of the control blank relative to itself (0 when there is no background interference)
• Dbt is displacement of test material in assay broth plus bacteria relative to the control
• Dt is displacement of the test material in assay broth relative to the control blank.
• the untreated "0" time control is represented by a solid line the one hour untreated control by a dot broken line ( . . . . ), the
• Figures 2a through 2d show the DLS response patterns for strains 6 (rec 34) and 11 (168 wild type) respectively. From the mean Y values of the "0" time and 1 hour samples, it is clear that increases in cell counts occurred in all untreated controls. From the mean Y values of the "0" time and 1 hour samples, it is clear that increases in cell counts occurred in all untreated controls. From the mean Y values of the "0" time and 1 hour samples, it is clear that increases in cell counts occurred in all untreated controls. From
• Figures 3a and 3b show the DLS response patterns for strains 6 (rec E4) and 11 (168 wild type) respectively. From the mean Y values of the "0" time and 1 hour samples, it is clear that increases in cell counts occurred in all untreated controls. From Figure 3a, it is clear that 3.6 ug/ml of MNNG caused a decreased arc length and a curve shift to the left, relative to the light scattering angles of the negative time "0" control. The relative intensity increased less than did the negative control from the "0" to 1 hour interval. from Figure 3b, it is clear that 3.6 ug/ml of MNNG did not cause a decrease in arc length.
• strains 6 and 11 were -3 and 19, respectively.
• Figures 5a through 5d show the DLS response patterns for strains 10 (FH2006-7) and 11 (168 wild type). From the mean Y values of the "0" time and one hour samples, it is clear that increases in cell counts occurred in all untreated controls. From Figures 5a and 5b, it is clear that 107 ug/ml and 53.6 ug/ml of
• Strain 10 at 107 and 5 3.6 ug/ml had PRI values of 222 and 131, respectively. At the 107 ug/ml level, a toxic and a sustained effect (cell enlargement) was seen. However, at the 53.6 ug/ml level, the bacteria recovered after 60 minutes. Strain 11 had PRI values of 131 at both concentrations. This indicated that at both levels, cell lethality and enlargement occurred but that full recovery resulting in growth and the return to normal size occurred by one hour. Although strain 10 may be slightly more affected than strain 11 at the higher concentration of Lindane, the differences in the PRI values do not appear to be useful in making toxicity assessments. The SRI values, however, were substantially different for strains 10 and 11.
• the SRI values for strain 10 at 107 ug/ml and 53.6 ug/ml were -19 and 7, respectively.
• the SRI value was 10 for strain 11 at both 107 ug/ml and 53.6 ug/ml.
• the dose-related SRI trend seen in strain 10 but not in strain 11 indicates a genotoxic effect of this chemical, as demonstrated by the negative SRI value at the higher dose level.
• strain 11 at both concentrations was capable of repairing genetic damage and dividing normally.
• Strain 10 was capable of repairing genetic damage and dividing normally only at the lower concentration of 53.6 ug/ml. From these results, it is clear that primary cytotoxicity may be expressed by a three digit number.
• the present invention is directed to the field of bioassays, and in particular to an assay utilizing radiation, or laser, technology in combination with unique genetically engineered bacteria that provide a
• sample preparation can be complex and different assays must be developed for the identification and quantification of individual chemicals or chemical classes.
• knowledge that a chemical is present and its concentration does not indicate its cytotoxic or genotoxic activity.
• the most widely employed bacterial test system for carcinogens is the Ames test, Ames, Mutat. Res. 31:347- 349 (1974), using Salmonella typhimurium histidine-requiring auxotrophs. Because the Ames assay relies on reverse mutation, a mutant deoxyribonucleic acid (DNA) locus must be mutated back to its wild-type configuration or be suppressed. Thus, chemical mutagens that cannot effect this change in the DNA will go undetected.
• researchers have recognized other shortcomings in this test, specifically an inability to devise an in vitro activation system that is reliable and reproducible, and an inability to detect some mutational events that can lead to cancer. Felkner, Microbial Testers: Probing Chemical Carcinogenesis, 177 (1981).
• Felkner discovered that ordinary transforming strains of Bacillus subtilis recovered from short exposures to the potent mutagenic and carcinogenic agent, 4-nitroquinoline-1-oxide (4NQO), while other strains incapable of recombination and/or dark repair did not.
• the ability or inability to recover was found to be directly correlated with the blocking of de novo deoxyribonucleic (DNA) synthesis through complexation of 4NQ0 with the DNA of the cell.
• DNA de novo deoxyribonucleic
• the product of this research was the discovery that a certain chemical structure has a corresponding particular biological function. Based on this principle, the effect of a chemical on a biological system, such as Bacillus subtilis bacteria, may be identified by the change in morphology of the assayed cells.
• Felkner developed a microbial bioassay from a collection of several hundred Bacillus subtilis mutants based on the principles derived from his earlier work. These selected strains had uniqu genetic defects causing them to be very sensitive to the effects of specific chemical classes, toxins and radiation. To enable uniformity, the mutant genes from these strains were introduced by recombination technology into identical clones of the wild type (normal) Bacillis subtilis strain. Thus, a battery of strains, differing only by a single character (gene) or limited set of genes was produced. The member strains are therefore isogenic, that is, identical except for single defined genes.
• Bioassays employing bacteria are disclosed in Wyatt U.S. Patent No. 3,730,842, issued May 1, 1973, and Wyatt U.S. Patent No. 4,101,383, issued July 18, 1978. These patents disclose methods of determining bacterial sensitivity or susceptibility to antibiotics by using test differential scattering patterns derived from the test bacteria and then comparing them to patterns of control bacteria. Neither reference provides for generating a pattern to screen for a toxicant having potential cytotoxic or genotoxic activity or to provide a means by which the toxicant may be identified and quantified. Wyatt U.S. patent No.
• 4,541,719 issued Septembe 17, 1985, discloses a laser apparatus that measure scattered radiation by microparticles such as bacteria at two well-defined scattering angles. The toxic response of bacteria is measured by averaging mean scattered intensities.
• Other patents naming Wyatt as an inventor include Wyatt U.S. Patent No. 4,693,602; Phillips U.S. Patent No. 4,616,927; Wyatt U.S. patent No. 4,621,063; Wyatt U.S. Patent No. 4,548,500; Wyatt U.S. Patent No. 4,490,042; Wyatt U.S. Patent No. 4,173,415; Wyatt U.S. Patent No. 3,928,140; Wyatt U.S. Patent No. 3,770,351; Wyatt U.S. Patent No. 3,754,830; and Wyatt U.S. Patent No. 3,624,835, which are directed to various methods and apparatus for characterizing microparticles.
• Another object of this invention is to provide unique differential light scattering (DLS) patterns for chemical agents using isogenic repair mutant and wild type strains of Bacillus subtilis.
• DLS differential light scattering
• a further object of this invention is to select from the Bacillus subtilis strains a minimum group or
• This invention has as yet another object the provision of a means to score the toxicity responses of the selected bacteria and to compare these responses to the responses of these bacteria to known chemical agents.
• Another object of this invention is to establish means for evaluating toxicity responses for compounds whose genotoxic responses have been difficult or even impossible to detect with other state-of-the-art mutagenicity assays.
• This invention has the further object to provide, as a reference, DLS patterns generated when these bacteria are treated with identified chemicals, to screen for toxicity and to identify and quantify samples.
• a further object of this invention is the provision of a medium for solubilizing water insoluble samples to be tested, this medium being harmless to the assay bacteria and being optically clear when mixed with the bacteria so that radiation may readily penetrate, and scattering may be detected.
• Bacillus subtilis isogenic strains of bacteria lacking certain genetic repair functions and strains that are repair efficient that can be monitored for their unique responses to toxic chemicals using a differential light-scattering laser system. These responses are then scored to determine, for example, acute toxic or genotoxic effects. These scores, calculated as response indices, are then compared to known responses of these bacteria strains to identified chemical agents. It is to be understood that this method is also capable of assessing quantifiably the presence of a chemical to be tested.
• Figure 1 is a schematic view of the method and apparatus of the claimed invention.
• Figures 2a through 2d are differential light scattering patterns generated when the assay bacteria is exposed to certain concentrations of 4-nitroquinoline-N-oxide (“4NQO").
• Figures 3a and 3b are differential light scattering patterns generated when the assay bacteria is exposed to certain concentrations of N-methyl-N'-nitro-N-nitroso-quanadine ( "MNNG” ).
• Figures 4a through 4d are differential light scattering patterns generated when the assay bacteria is exposed to certain concentrations of benzo(a)pyrene (“B(a)P").
• Figures 5a through 5d are differential light scattering patterns generated when the assay bacteria is exposed to certain concentrations of "Lindane”.
• FIG. 1 provides a schematic illustration of the claimed apparatus by which samples are bioassayed in accord with the claimed invention.
• the bioassay consists of a radiation source 4, such as a laser system, which in the preferred embodiment employs the laser system such as that disclosed in Wyatt U.S. Patent No. 4,541,719, which includes a laser producing monochromatic visible or infrared radiation that is plane polarized with respect to two arrays of detectors.
• the laser system is capable of making 1200 measurements/second at 15 unique angles.
• the bioassay further includes an isogenic set of Bacillus subtilis mutants 2. The laser illuminates the cuvette 1 containing the bacteria 2 in suspension, these bacteria causing the light to be differentially scattered.
• the differential light scattering (DLS) 6 of the laser beam is detected as scattered light intensity as a function of angle.
• the detectors 5 detect the DLS and feed the signals to a recorder 7, which records, in the form of a graph, a plot of the scattered intensity.
• an analysis apparatus such as a computer 8 receive the data from the detectors 5 directly.
• the intensity variation in the scattered light is called the DLS pattern, and is dependent on the average size and structure of the bacteria as well as the population size distribution.
• the DLS pattern of a suspension shows how many particles are present, their size, shape and the distribution of particles.
• Bacterial count is expressed as a measure of height, or the highest point, of the DLS pattern.
• the shift of peaks of a sample DLS pattern indicates the degree of morphological change as compared to the control pattern.
• the DLS pattern may be in magnetically recorded digital form for machine readability, or recorded in graphic form such that the scattered intensity pattern is represented as an eye readable curve.
• a digitizing tablet (not shown) is used to trace each pattern's curve and simultaneously transmit a continuous stream of x and y coordinates to an analysis apparatus such as a computer 8.
• an analysis apparatus such as a computer 8.
• the analysis apparatus which is in the preferred embodiment a computer 8 carries out the required calculations of response indices representing the shift in the DLS of the control as compared to the sample, and the change in bacterial count.
• the derived indices provide an immediate preliminary indication of toxic response, the DNA damaging (genotoxic) potential and aid in the identification of "isosets" of the Bacillus subtilis bacteria for compound identification.
• the concentration of the chemical sample is determined by the level of response by the bacteria to the given sample.
• the set of bacteria selected for the bioassay each differ by only one property, which causes them to be more sensitive than other set members because of the mechanism of toxicity. Therefore, a "fingerprint", or profile unique to each chemical is generated from the differential response of the member bacteria. By "scoring" these fingerprints, the bioassay provides data for identifying test chemicals, assessing the concentration of test chemicals, and assessing whether the test chemical is acutely or chronically toxic.
• Another embodiment of the invention provides a library 9 wherein are stored the DLS patterns and calculated response indices for the bioassay of identified samples. By comparing the sample DLS with the stored pattern, the sample may be screened for toxicity, identified, and/or quantified.
• the time required for a complete assay is approximately sixty (60) to sixty-six (66) minutes, the time required for untreated bacteria to divide twice in the absence of any chemical sample. However, individual samples may be analyzed at a rate of one sample per minute to determine the concentrations of a chemical in an aqueous sample.
• the Bacteria The bacteria used in the bioassay are of a single species, Bacillus subtilis, which has numerous advantages over other bacteria traditionally used in bioassays. First, Bacillus subtilis is the most widely studied and genetically mapped gram-positive microorganism. Henner, D.J. and Hoch, J.A., The Bacillus subtilis Chromosome, Microbiol. Rev. 44:57-82 (1980).
• Bacillus subtilis exists as a spore that is capable of surviving genetically unchanged while resisting heat, drying or other adverse effects for an indefinite period.
• the bacteria can divide rapidly to double itself in approximately twenty-six (26) minutes during the logarithmic growth phase, and is much more sensitive at this stage to chemical toxicants than are the gram-negative microorganisms, Salmonella typhimurium and Escherichia coli.
• Bacillus subtilis readily takes up large molecules such as DNA, and can integrate genes derived from members of the same species or other species through traditional recombinant DNA technology.
• the bioassay uses Bacillus subtilis mutants that are genetically identical, or isogenic, except for one or more genetic blocks in unique enzymatic repair processes or steps that restore chemically damaged DNA to its functional condition.
• Bacillus subtilis are specifically selected strains deficient in different recombination (Rec-), excision (exc-), polymerase (Pol-) or spore repair (Spp-) repair steps.
• These strains may be deficient in one or a multiple of repair steps, and include the Rec- strains, recA1, recA8, recB2, recC5, recD3, recE4, recG13, mc-1, and m45; the Pol- strains T-1, TKJ8201; the Exc- strains her-9 and TKJ8206; The Exc- and Rec- strain FH2006-7; the Exc-, Rec- and Pol- strain HJ15; the Exc- and Spp- strain TKJ5211; the Exc-, Pol- and Spp- strain TKJ6321; and repair efficient strains HA101, 168 and 168 wild type.
• the mutants in this group are therefore more sensitive to the metabolic toxicity or genotoxic activity or a broad range of chemical substances at nannomolar or nannogram concentrations.
• Bacillus subtilis mutants were developed by treating them with by irradiation or with various chemical mutagens. For example, nitrosoquanidine, a powerful mutagenic chemical, was used to change the genetic makeup of the bacteria and trimethoprim to select for mutant types that could not synthesize DNA unless thymine was provided to them.
• the mutants from one group isolated by Felkner were designated FH2001 through FH2006 and had been derived from a parent strain JB01-200 whose lineage is defined in Felkner J. Bacteriol. 104:1030-1032 (1970).
• trp+, thy+, her-, rec- Types 2 and 3 are mutants suitable for assaying her- and rec- mutations to detect DNA damage. These bacteria, constructed by conventional genetic engineering techniques would not occur in nature or result in a surviving bacterial isolate.
• recA8, recA1, recB2, recC5, recD3, recE4, recG13, her-9, mc-1 and FH2006-7 are descended from strain 168. This strain is a descendent from the strain trp C of John Spizizen. " Spizizen, J. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate, Proc. Nat. Acad. Sci.
• Isosets defined as a subset of these mutants, constitute the minimum number required to define a response to a sample tested.
• Data derived form fractional survival tests and data derived from spot assays are used to determine which B. subtilis strains should be selected to comprise an isoset to identify a given compound by the radiation bioassay.
• the isogenic mutants are pre-screened using the spot test with given chemical samples.
• the DNA-damaging test is performed according to the procedure disclosed in Felkner, I.C., Microbial Testers: Probing Carcinogenesis (1981) and Felkner, I.C. in Medine, A., and Anderson, W., eds. Proceedings of the National Conference on Environmental Engineering, Am. Soc. Civil Eng., N.Y. pp. 204-209 (1983).
• Each mutant is inoculated into 5 ml of Brain Heart Infusion (BHI) broth, either from a disc containing approximately 1 ⁇ 10 7 washed spores or a 1 mm loopful from a sporulating slant culture, and incubated by shaking at 37 to 39° C for 16 hours.
• This culture is designated o/n.
• inocula from the o/n cultures are streaked radially on a nutrient agar plate to a sensitivity disc containing 10 ul of the assay chemical which may or may not have been pre-incubated with a rat liver microsomal fraction (S-9) in order to achieve metabolic activation.
• Liver microsomes designated an S-9 fraction, were prepared from Sprague-Dawley rats induced with Aroclor 1254 (Litton Bionetics, Washington, SC 19405). The S-9 fraction was prepared as a KCl homogenate with a protein concentration of 25-28 mg/ml. It is understood that this fraction may be microsomal or post-microsomal and is not limited by the origin of the tissues.
• a "cocktail” was therefore derived so that a clear aqueous solution of the test chemical could be mixed with the assay bacteria and the hydrophilic S-9 fraction, thereby minimizing interference with the radiation source and its ability to generate a DLS pattern of the radiation scattered by said bacteria.
• the "cocktail” has the advantage of solubilizing chemicals in an aqueous system that were not as readily solubilized by other known procedures using dimethylsulfoxide (DMSO), ethanol or acetone before being introduced into the aqueous assay mixture.
• DMSO dimethylsulfoxide
• acetone acetone
• the preferred embodiment of the bioassay employs a dispersant sold under the trademark ".COREXIT 7664 Oil Dispersant” (Exxon Chemicals, Clark, New Jersey) and a non-ionic surfactant sold under the trademark “EMULPHOR EL-620” (GAF Corp., 140 West 51 Street, New York, N.Y. 10020). Both the "COREXIT” dispersant and the “EMULPHOR” surfactant are readily obtainable at the present time, and are generally known to chemists and commercial users. The chemical compositions of each of these are maintained as trade secrets by the respective owner companies.
• the "EMULPHOR” surfactant is a polyoxylated vegetable oil.
• the radiation source 1 of the bioassay is a laser such as that disclosed in Wyatt, U.S. Patent No. 4,541,719. This instrument records scattering intensity at fifteen angular locations, permitting the relationships between refractive index, size and shape to be measured. Moreover the read head is anodized, sealed and attached directly to the laser head as well as to an integrated circuit board, providing a unitized optical bench and a grounded enclosure for the detector elements.
• Computer interface Parallel analog outputs interfaced to 16-channel, 12 bit A/D programmable multiplexer.
• Detectors Transimpedance photodiodes with built-in amplifiers Detector Output Dynamic Range: 0 - 10 V Dynamic Range: 10 4 Linearity: ⁇ 0.1%
• the chemical B(a)P is a well known procarcinogen/mutagen that is insoluble in an aqueous system, and requires metabolic activation by rat liver microsomal fractions (S-9) to become electrophilic and to cause DNA damage or a mutagenic response.
• "Lindane” is a chemical difficult to assay for its genotoxic potential by state of the art mutagenic assays.
• Lyphilized tablets * containing the appropriate Bacillus subtilis strains and dehydrated growth medium are introduced into cuvettes containing water. Suspensions of bacterial cells at the required concentration described in the examples provided infra may also be used.
• the inoculated cuvettes are placed in an incubation chamber that has been prewarmed to approximately 37 to 39° C, and incubation continued for about one hour to one and a half hours to establish a culture in the "assay-ready condition".
• Test or control material is introduced into the cuvettes using one pair of cuvettes for each assay organism.
• a negative control having no test material, and a positive control having an identified toxic chemical at a concentration whose response has been determined is included.
• the cuvettes are incubated for six minutes at 37-39° C, and each cuvette is read a minimum of fifty
• the bacteria could be lyphilized vegetative cells or spores in a capsule form. However, when spores are used, this form being preferred because of their stability, they must be heat-shocked at 60 degrees
• Centigrade Include a negative control having no test material, and a positive control having a toxic chemical at a concentration whose response has been determined.
• a negative control may also comprise a control sample of bacteria exposed to 4-nitroquinoline-N-oxide or 4-nitroquinoline-1-oxide and a positive control of bacteria exposed to benzo(a)pyrene.
• Cultivation of bacterial cells appropriate in concentration for the bioassay includes heat shocking strains of Bacillus subtilis in 1 ml of Brain Heart Infusion (BHI) broth for 20 minutes. Additional broth is added until an absorbance of 0.2 A(540) is achieved. The cultures are incubated at 37 to 39° C until the bacterial growth reaches an A(540) of 0.5, a period of approximately 2 hours.
• Cultures that require a longer period to reach this density are not considered suitable for the bioassay. Cultures found suitable are diluted to an A(540) of 0.3 with BHI broth yielding approximately 2 ⁇ 10 7 bacteria/ml. These cells, when diluted by the amount required for the cuvette assay, yield a cell concentration of 10 6 bacteria/ml.
• 4NQO was assayed at final concentrations of 8.5, 4.2 and 2.1 ug/ml using Bacillus subtilis strains recE4 (6), 168 or 168 wild type (11).
• the DLS patterns for each of these assays comprise Figures 2a through 2d.
• MNNG was assayed at a final concentration of 3.6 ug/ml with strains 6 and 11.
• the DLS patterns for each of these assays comprise Figures 3a and b.
• a screw cap tube containing 13.6 ml of deionized water at 37 to 39° C is pipetted 0.3 ml of bacteria and 0.1 ml of the test chemical.
• the tube is inverted gently and its contents poured into a cuvette.
• the cuvette is illuminated by the laser and the intensities of scattered light measured and scored.
• the cuvette is then incubated at 37-39 degrees
• the method may also be performed using lyphilized bacteria having with it sufficient dehydrated BHI to support the growth during the assay period.
• the total volume of fluid required to obtain a reading from the cuvette is 10 ml. Therefore, a volume of about 0.4 ml of fluid will rehydrate the cell-medium.
• the incubation period remains unchanged, and illuminating is performed at fifteen minute intervals.
• B(a)P was assayed at final concentrations of 15.1 and 7.6 ug/ml using Bacillus subtilis strains 11 and TKJ6321 (19).
• B(a)P was assayed at final concentrations of 15.1 and 7.6 ug/ml using Bacillus subtilis strains 11 and TKJ6321 (19).
• the DLS patterns generated by this assay comprise Figures 4a-4d.
• the aqueous cocktail of "COREXIT”, "EMULPHOR” and water is prepared prior to addition of the S-9 fraction and the bacteria.
• 180 ul each of "EMULPHOR” and “COREXIT” are added to each 5 ml of deionized water and mixed.
• 180 ul of S-9 fraction is added along with appropriate amount of lyphilized bacteria and medium required to achieve a cell concentration of 10 6 bacteria/ml.
• the scoring techniques of the bioassay measure the height of the DLS pattern as a function of bacterial count and the amount of shift in the peaks of the chemical sample pattern and the control sample pattern to determine the degree of morphological change in size and shape of the bacteria.
• the derived indices provide an immediate preliminary indication of the toxic response, the DNA damaging (genotoxic) potential, and identify isosets for compound identification.
• each pattern's height provided an indication of bacterial count and is therefore the principal indicator of toxicity.
• scoring techniques for the identification of morphological changes it was noted that there is a function which is continuous on a closed interval from the beginning of the pattern to its end. Each line segment between any two adjacent points formed an arc, and associated with each point on the X-axis is only one point on the pattern. Using a digitizing tablet, it is possible to obtain a sufficiently large number of points so that the sum of the distances between all adjacent points would approximate the length of the pattern. This was defined as the arc length of the curve. Variances of the arc length correspond with the variances in the bacteria morphology, especially as they related to individual cell size.
• the percent change between the test pattern's arc length and the baseline pattern's arc length was calculated. To determine the fourth order arc index, the arc percent change was divided by 5 and, if the change was positive, 1 was added. If the change was negative, 2 was subtracted. The integer value was retained.
• Each of the three digits, from left to right in the PRI index, derives from the following table according to the fourth order indices of the patterns, starting with the control at "0" time as the reference and the control at 60 minutes as the first digit.
• the tertiary responses for each of the indexed patterns were obtained by multiplying the fourth order Y index by the fourth order arc index.
• the secondary response index (SRI) was determined by the calculation of the sum of the tertiary indices for all indexed patterns combined. This method of calculation is illustrated by the following data derived from an assay:
• the interpretation of the PRI of 132 is that the control of sixty-six (66) minutes displayed normal growth, hence the number 1 as the first digit.
• the treated sample at time "0" showed a loss in bacterial count accompanied by size increase, hence the number 3 as the second digit.
• sixty-six minutes the treated sample showed an increase in bacterial count accompanied by a size increase, hence the number 2 in the third digit.
• PRIs Two extreme examples of PRIs are 133 and 144. These examples are immediately toxic responses from which the cells fail, to recover.
• the 133 PRI shows cell enlargement, and the 144 PRI shows cell shrinkage, both being associated with the failure of the cell to recover but by different mechanisms of action.
• the presence of a 3 or 4 in the first digit of any PRI should mean that the assay is invalid because the bacterial control culture is not displaying normal growth.
• a screening score S provides a precise measure of bacterial growth or inhibition by bacteria in the test sample as compared to the control bacteria. It is defined in terms of DLS placement scores and was implemented by previous studies. Wyatt, P.J., Phillips, E.T., Scher, M.G., Kahn, M.R., and Allen, E.H., J. Argic. Food Chem. 25:1080 (1977); Wyatt, P.J., Scher, M.G., and Phillips, D.T., J. Agric. Food Chem. 25:1086 (1977). Thus,
• Dbc is the DLS displacement of the assay broth plus bacteria relative to the assay broth (control)
• Dc is the displacement of the control blank relative to itself (0 when there is no background interference)
• Dbt is displacement of test material in assay broth plus bacteria relative to the control
• Dt is displacement of the test material in assay broth relative to the control blank.
• the untreated "0" time control is represented by a solid line the one hour untreated control by a dot broken line ( . . . . ), the "0" time treated sample by a widely spaced broken line ( - - - - ), and the one hour treated sample by a narrow spaced broken line (_ _ _ _ ).
• Figures 2a through 2d show the DLS response patterns for strains 6 (rec 34) and 11 (168 wild type) respectively. From the mean Y values of the "0" time and 1 hour samples, it is clear that increases in cell counts occurred in all untreated controls. From Figures 2a and 2b, it is clear that 4.2 ug/ml and 2.1 ug/ml of 4NQ0 caused increased arc lengths and curves shifting to the left relative to the light scattering angles of the negative time "0" control. The relative intensity increased less from the "0" to 1 hour interval.
• Figures 3a and 3b show the DLS response patterns for strains 6 (rec E4 ) and 11 (168 wild type) respectively. From the mean Y values of the "0" time and 1 hour samples, it is clear that increases in cell counts occurred in all untreated controls. From Figure 3a, it is clear that 3.6 ug/ml of MNNG caused a decreased arc length and a curve shift to the left, relative to the light scattering angles of the negative time "0" control. The relative intensity increased less than did the negative control from the "0" to 1 hour interval.
• Strain 11 at the concentration of 3.6 ug/ml had a PRI value of 111, thus showing no compound-related toxicity expressed either immediately or over the 60-minute period following treatment. This is a genotoxic response expressed at the primary response level. Additionally, the SRI value for strains 6 and 11 were -3 and 19, respectively. The negative secondary response of strain 6, when compared to the positive SRI of strain 11, indicates that there is a genotoxic effect which can be repaired by the wild type (strain 11) but not the Rec- mutant (strain 6). Lindane DLS Toxicity Responses:
• Figures 5a through 5d show the DLS response patterns for strains 10 (FH2006-7) and 11 (168 wild type). From the mean Y values of the "0" time and one hour samples, it is clear that increases in cell counts occurred in all untreated controls. From Figures 5a and 5b, it is clear that 107 ug/ml and 53.6 ug/ml of Lindane initially caused decreased arc lengths and curves shifting to the left relative to the light scattering angles of the negative time "0" control. However, at the lower level, a positive shift was seen at sixty minutes. The relative intensity did not change substantially from the "0" to one hour intervals at the higher level. However, an increase was seen at the lower concentration.
• strain 11 had PRI values of 131 at both concentrations. This indicated that at both levels, cell lethality and enlargement occurred but that full recovery resulting in growth and the return to normal size occurred by one hour.
• strain 10 may be slightly more affected than strain 11 at the higher concentration of Lindane, the differences in the PRI values do not appear to be useful in making toxicity assessments.
• the SRI values were substantially different for strains 10 and 11.
• the SRI values for strain 10 at 107 ug/ml and 53.6 ug/ml were -19 and 7, respectively.
• the SRI value was 10 for strain 11 at both 107 ug/ml and 53.6 ug/ml.
• strain 11 at both concentrations, was capable of repairing genetic damage and dividing normally.
• Strain 10 was capable of repairing genetic damage and dividing normally only at the lower concentration of 53.6 ug/ml. From these results, it is clear that primary cytotoxicity may be expressed by a three digit number. Using the primary response of mutant and wild type bacteria, some compounds such as MNNG and 4NPO which are highly toxic can also be identified as genotoxic.
• the present invention is directed to the field of bioassays, and in particular to an assay utilizing radiation, or laser, technology in combination with unique genetically engineered bacteria that provide a "fingerprinting" capacity to identify and measure concentrations of toxicants in various environmental media.
• sample preparation can be complex and different assays must be developed for the identification and quantification of individual chemicals or chemical classes.
• knowledge that a chemical is present and its concentration does not indicate its cytotoxic or genotoxic activity.
• the most widely employed bacterial test system for carcinogens is the Ames test, Ames, Mutat. Res. 31:347- 349 (1974), using Salmonella typhimurium histidine- requiring auxotrophs. Because the Ames assay relies on reverse mutation, a mutant deoxyribonucleic acid (DNA) locus must be mutated back to its wild-type configuration or be suppressed. Thus, chemical mutagens that cannot effect this change in the DNA will go undetected.
• researchers have recognized other shortcomings in this test, specifically an inability to devise an in vitro activation system that is reliable and reproducible, and an inability to detect some mutational events that can lead to cancer. Felkner, Microbial Testers: Probing Chemical Carcinogenesis, 177 (1981).
• Felkner discovered that ordinary transforming strains of Bacillus subtilis recovered from short exposures to the potent mutagenic and carcinogenic agent, 4-nitroquinoline-1-oxide (4NQO), while other strains incapable of recombination and/or dark repair did not.
• the ability or inability to recover was found to be directly correlated with the blocking of de novo deoxyribonucleic (DNA) synthesis through complexation of 4NQO with the DNA of the cell.
• DNA de novo deoxyribonucleic
• the product of this research was the discovery that a certain chemical structure has a corresponding particular biological function. Based on this principle, the effect of a chemical on a biological system, such as Bacillus subtilis bacteria, may be identified by the change in morphology of the assayed cells.
• Felkner developed a microbial bioassay from a collection of several hundred Bacillus subtilis mutants based on the principles derived from his earlier work. These selected strains had unique genetic defects causing them to be very sensitive to the effects of specific chemical classes, toxins and radiation. To enable uniformity, the mutant genes from these strains were introduced by recombination technology into identical clones of the wild type (normal) Bacillis subtilis strain. Thus, a battery of strains, differing only by a single character (gene) or limited set of genes was produced. The member strains are therefore isogenic, that is, identical except for single defined genes.
• Bioassays employing bacteria are disclosed in Wyatt U.S. Patent No. 3,730,842, issued May 1, 1973, and Wyatt U.S. Patent No. 4,101,383, issued July 18, 1978. These patents disclose methods of determining bacterial sensitivity or susceptibility to antibiotics by using test differential scattering patterns derived from the test bacteria and then comparing them to patterns of control bacteria. Neither reference provides for generating a pattern to screen for a toxicant having potential cytotoxic or genotoxic activity or to provide a means by which the toxicant may be identified and quantified.
• Wyatt U.S. patent No. 4,541,719, issued September 17, 1985 discloses a laser apparatus that measures scattered radiation by microparticles such as bacteria at two well-defined scattering angles.
• Another object of this invention is to provide unique differential light scattering (DLS) patterns for chemical agents using isogenic repair mutant and wild type strains of Bacillus subtilis.
• DLS differential light scattering
• a further object of this invention is to select from the Bacillus subtilis strains a minimum group or "isoset" required to establish response patterns to specific chemical classes.
• This invention has as yet another object the provision of a means to score the toxicity responses of the selected bacteria and to compare these responses to the responses of these bacteria to known chemical agents.
• Another object of this invention is to establish means for evaluating toxicity responses for compounds whose genotoxic responses have been difficult or even impossible to detect with other state-of-the-art mutagenicity assays.
• This invention has the further object to provide, as a reference, DLS patterns generated when these bacteria are treated with identified chemicals, to screen for toxicity and to identify and quantify samples.
• a further object of this invention is the provision of a medium for solubilizing water insoluble samples to be tested, this medium being harmless to the assay bacteria and being optically clear when mixed with the bacteria so that radiation may readily penetrate, and scattering may be detected.
• the foregoing and other objects of the invention are achieved by the provision of Bacillus subtilis isogenic strains of bacteria lacking certain genetic repair functions and strains that are repair efficient that can be monitored for their unique responses to toxic chemicals using a differential light-scattering laser system. These responses are then scored to determine, for example, acute toxic or genotoxic effects. These scores, calculated as response indices, are then compared to known responses of these bacteria strains to identified chemical agents. It is to be understood that this method is also capable of assessing quantifiably the presence of a chemical to be tested.
• Figure 1 is a schematic view of the method and apparatus of the claimed invention.
• Figures 2a through 2d are differential light scattering patterns generated when the assay bacteria is exposed to certain concentrations of 4-nitroquinoline-N-oxide (“4NQO").
• Figures 3a and 3b are differential light scattering patterns generated when the assay bacteria is exposed to certain concentrations of N-methyl-N'-nitro-N-nitroso-quanadine ( "MNNG").
• MNNG N-methyl-N'-nitro-N-nitroso-quanadine
• Figures 4a through 4d are differential light scattering patterns generated when the assay bacteria is exposed to certain concentrations of benzo(a)pyrene ("B(a)P").
• Figures 5a through 5d are differential light scattering patterns generated when the assay bacteria is exposed to certain concentrations of "Lindane”.
• FIG. 1 provides a schematic illustration of the claimed apparatus by which samples are bioassayed in accord with the claimed invention.
• the bioassay consists of a radiation source 4, such as a laser system, which in the preferred embodiment employs the laser system such as that disclosed in Wyatt U.S. Patent No. 4,541,719, which includes a laser producing monochromatic visible or infrared radiation that is plane polarized with respect to two arrays of detectors.
• the laser system is capable of making 1200 measurements/second at 15 unique angles.
• the bioassay further includes an isogenic set of Bacillus subtilis mutants 2. The laser illuminates the cuvette 1 containing the bacteria 2 in suspension, these bacteria causing the light to be differentially scattered.
• the differential light scattering (DLS) 6 of the laser beam is detected as scattered light intensity as a function of angle.
• the detectors 5 detect the DLS and feed the signals to a recorder 7, which records, in the form of a graph, a plot of the scattered intensity.
• an analysis apparatus such as a computer 8 receive the data from the detectors 5 directly.
• the intensity variation in the scattered light is called the DLS pattern, and is dependent on the average size and structure of the bacteria as well as the population size distribution.
• the DLS pattern of a suspension shows how many particles are present, their size, shape and the distribution of particles.
• Bacterial count is expressed as a measure of height, or the highest point, of the DLS pattern.
• the shift of peaks of a sample DLS pattern indicates the degree of morphological change as compared to the control pattern.
• the DLS pattern may be in magnetically recorded digital form for machine readability, or recorded in graphic form such that the scattered intensity pattern is represented as an eye readable curve.
• a digitizing tablet (not shown) is used to trace each pattern's curve and simultaneously transmit a continuous stream of x and y coordinates to an analysis apparatus such as a computer 8.
• an analysis apparatus such as a computer 8.
• the analysis apparatus which is in the preferred embodiment a computer 8 carries out the required calculations of response indices representing the shift in the DLS of the control as compared to the sample, and the change in bacterial count.
• the derived indices provide an immediate preliminary indication of toxic response, the DNA damaging (genotoxic) potential and aid in the identification of "isosets" of the Bacillus subtilis bacteria for compound identification.
• the concentration of the chemical sample is determined by the level of response by the bacteria to the given sample.
• the set of bacteria selected for the bioassay each differ by only one property, which causes them to be more sensitive than other set members because of the mechanism of toxicity. Therefore, a "fingerprint", or profile unique to each chemical is generated from the differential response of the member bacteria. By "scoring" these fingerprints, the bioassay provides data for identifying test chemicals, assessing the concentration of test chemicals, and assessing whether the test chemical is acutely or chronically toxic.
• Another embodiment of the invention provides a library 9 wherein are stored the DLS patterns and calculated response indices for the bioassay of identified samples. By comparing the sample DLS with the stored pattern, the sample may be screened for toxicity, identified, and/or quantified.
• the time required for a complete assay is approximately sixty (60) to sixty-six (66) minutes, the time required for untreated bacteria to divide twice in the absence of any chemical sample. However, individual samples may be analyzed at a rate of one sample per minute to determine the concentrations of a chemical in an aqueous sample.
• the Bacteria The bacteria used in the bioassay are of a single species, Bacillus subtilis, which has numerous advantages over other bacteria traditionally used in bioassays. First, Bacillus subtilis is the most widely studied and genetically mapped gram-positive microorganism. Henner, D.J. and Hoch, J.A., The Bacillus subtilis Chromosome, Microbiol. Rev. 44:57-82 (1980).
• Bacillus subtilis exists as a spore that is capable of surviving genetically unchanged while resisting heat, drying or other adverse effects for an indefinite period.
• the bacteria can divide rapidly to double itself in approximately twenty-six (26) minutes during the logarithmic growth phase, and is much more sensitive at this stage to chemical toxicants than are the gram-negative microorganisms, Salmonella typhimurium and Escherichia coli.
• Bacillus subtilis readily takes up large molecules such as DNA, and can integrate genes derived from members of the same species or other species through traditional recombinant DNA technology.
• the bioassay uses Bacillus subtilis mutants that are genetically identical, or isogenic, except for one or more genetic blocks in unique enzymatic repair processes or steps that restore chemically damaged DNA to its functional condition.
• Bacillus subtilis are specifically selected strains deficient in different recombination (Rec-), excision (exc-), polymerase (Pol-) or spore repair (Spp-) repair steps.
• These strains may be deficient in one or a multiple of repair steps, and include the Rec- strains, recA1, recA8, recB2, recC5, recD3, recE4, recG13, mc-1, and m45; the Pol- strains T-1, TKJ8201; the Exc- strains her-9 and TKJ8206; The Exc- and Rec- strain FH2006-7; the Exc-, Rec- and Pol- strain HJ15; the Exc- and Spp- strain TKJ5211; the Exc-, Pol- and Spp- strain TKJ6321; and repair efficient strains HA101, 168 and 168 wild type.
• the mutants in this group are therefore more sensitive to the metabolic toxicity or genotoxic activity or a broad range of chemical substances at nannomolar or nannogram concentrations.
• Bacillus subtilis mutants were developed by treating them with by irradiation or with various chemical mutagens. For example, nitrosoquanidine, a powerful mutagenic chemical, was used to change the genetic makeup of the bacteria and trimethoprim to select for mutant types that could not synthesize DNA unless thymine was provided to them.
• the mutants from one group isolated by Felkner were designated FH2001 through FH2006 and had been derived from a parent strain JB01-200 whose lineage is defined in Felkner J. Bacteriol. 104:1030-1032 (1970).
• trp+, thy+, her-, rec- Types 2 and 3 are mutants suitable for assaying her- and rec- mutations to detect DNA damage. These bacteria, constructed by conventional genetic engineering techniques would not occur in nature or result in a surviving bacterial isolate.
• recA8, recA1, recB2, recC5, recD3, recE4, recG13, hcr-9, mc-1 and FH2006-7 are descended from strain 168. This strain is a descendent from the strain trp C of John Spizizen. Spizizen, J. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate, Proc. Nat. Acad. Sci.
• Isosets defined as a subset of these mutants, constitute the minimum number required to define a response to a sample tested.
• Data derived form fractional survival tests and data derived from spot assays are used to determine which B. subtilis strains should be selected to comprise an isoset to identify a given compound by the radiation bioassay.
• the isogenic mutants are pre-screened using the spot test with given chemical samples.
• the DNA-damaging test is performed according to the procedure disclosed in Felkner, I.C., Microbial Testers: Probing Carcinogenesis (1981) and Felkner, I.C. in Medine, A., and Anderson, W. , eds. Proceedings of the National Conference on Environmental Engineering, Am. Soc. Civil Eng., N.Y. pp. 204-209 (1983).
• Each mutant is inoculated into 5 ml of Brain Heart Infusion (BHI) broth, either from a disc containing approximately 1 ⁇ 10 7 washed spores or a 1 mm loopful from a sporulating slant culture, and incubated by shaking at 37 to 39° C for 16 hours.
• This culture is designated o/n.
• inocula from the o/n cultures are streaked radially on a nutrient agar plate to a sensitivity disc containing 10 ul of the assay chemical which may or may not have been pre-incubated with a rat liver microsomal fraction (S-9) in order to achieve metabolic activation.
• Liver microsomes designated an S-9 fraction, were prepared from Sprague-Dawley rats induced with Aroclor 1254 (Litton Bionetics, Washington, SC 19405). The S-9 fraction was prepared as a KCl homogenate with a protein concentration of 25-28 mg/ml. It is understood that this fraction may be microsomal or post-microsomal and is not limited by the origin of the tissues.
• a "cocktail” was therefore derived so that a clear aqueous solution of the test chemical could be mixed with the assay bacteria and the hydrophilic S-9 fraction, thereby minimizing interference with the radiation source and its ability to generate a DLS pattern of the radiation scattered by said bacteria.
• the "cocktail” has the advantage of solubilizing chemicals in an aqueous system that were not as readily solubilized by other known procedures using dimethylsulfoxide (DMSO), ethanol or acetone before being introduced into the aqueous assay mixture.
• DMSO dimethylsulfoxide
• acetone acetone
• the preferred embodiment of the bioassay employs a dispersant sold under the trademark "COREXIT 7664 Oil Dispersant” (Exxon Chemicals, Clark, New Jersey) and a non-ionic surfactant sold under the trademark “EMULPHOR EL-620” (GAF Corp., 140 West 51 Street, New York, N.Y. 10020). Both the "COREXIT” dispersant and the “EMULPHOR” surfactant are readily obtainable at the present time, and are generally known to chemists and commercial users. The chemical compositions of each of these are maintained as trade secrets by the respective owner companies.
• the "EMULPHOR” surfactant is a polyoxylated vegetable oil.
• the radiation source 1 of the bioassay is a laser such as that disclosed in Wyatt, U.S. Patent No. 4,541,719. This instrument records scattering intensity at fifteen angular locations, permitting the relationships between refractive index, size and shape to be measured. Moreover the read head is anodized, sealed and attached directly to the laser head as well as to an integrated circuit board, providing a unitized optical bench and a grounded enclosure for the detector elements.
• Computer interface Parallel analog outputs interfaced to 16-channel, 12 bit A/D programmable multiplexer.
• Detectors Transimpedance photodiodes with built-in amplifiers Detector Output Dynamic Range: 0 - 10 V Dynamic Range: 10 4 Linearity: ⁇ 0.1%
• the chemical B(a)P is a well known procarcinogen/mutagen that is insoluble in an aqueous system, and requires metabolic activation by rat liver microsomal fractions (S-9) to become electrophilic and to cause DNA damage or a mutagenic response.
• "Lindane” is a chemical difficult to assay for its genotoxic potential by state of the art mutagenic assays.
• Lyphilized tablets containing the appropriate Bacillus subtilis strains and dehydrated growth medium are introduced into cuvettes containing water. Suspensions of bacterial cells at the required concentration described in the examples provided infra may also be used.
• the inoculated cuvettes are placed in an incubation chamber that has been prewarmed to approximately 37 to 39° C, and incubation continued for about one hour to one and a half hours to establish a culture in the "assay-ready condition" .
• Test or control material is introduced into the cuvettes using one pair of cuvettes for each assay organism.
• a negative control having no test material, and a positive control having an identified toxic chemical at a concentration whose response has been determined is included.
• the cuvettes are incubated for six minutes at 37-39° C, and each cuvette is read a minimum of fifty
• the bacteria could be lyphilized vegetative cells or spores in a capsule form. However, when spores are used, this form being preferred because of their stability, they must be heat-shocked at 60 degrees Centigrade for 8 to 10 minutes before incubation.
• An alternative protocol follows: 1. Solubilize a sample in an aqueous solution. For water insoluble samples, use a sufficient amount of "cocktail" to prevent precipitation of the sample.
• Centigrade Include a negative control having no test material, and a positive control having a toxic chemical at a concentration whose response has been determined.
• a negative control may also comprise a control sample of bacteria exposed to 4-nitroquinoline-N-oxide or 4-nitroquinoline-1-oxide and a positive control of bacteria exposed to benzo(a)pyrene.
• Cultivation of bacterial cells appropriate in concentration for the bioassay includes heat shocking strains of Bacillus subtilis in 1 ml of Brain Heart Infusion (BHI) broth for 20 minutes. Additional broth is added until an absorbance of 0.2 A(540) is achieved. The cultures are incubated at 37 to 39° C until the bacterial growth reaches an A( 540 ) of 0.5 , a period of approximately 2 hours.
• Cultures that require a longer period to reach this density are not considered suitable for the bioassay. Cultures found suitable are diluted to an A(540) of 0.3 with BHI broth yielding approximately 2 ⁇ 10 7 bacteria/ml. These cells, when diluted by the amount required for the cuvette assay, yield a cell concentration of 10 6 bacteria/ml.
• 4NQO was assayed at final concentrations of 8.5, 4.2 and 2.1 ug/ml using Bacillus subtilis strains recE4 (6), 168 or 168 wild type (11).
• the DLS patterns for each of these assays comprise Figures 2a through 2d.
• MNNG was assayed at a final concentration of 3.6 ug/ml with strains 6 and 11.
• the DLS patterns for each of these assays comprise Figures 3a and b.
• a screw cap tube containing 13.6 ml of deionized water at 37 to 39° C is pipetted 0.3 ml of bacteria and 0.1 ml of the test chemical.
• the tube is inverted gently and its contents poured into a cuvette.
• the cuvette is illuminated by the laser and the intensities of scattered light measured and scored.
• the cuvette is then incubated at 37-39 degrees
• the method may also be performed using lyphilized bacteria having with it sufficient dehydrated BHI to support the growth during the assay period.
• the total volume of fluid required to obtain a reading from the cuvette is 10 ml. Therefore, a volume of about 0.4 ml of fluid will rehydrate the cell-medium.
• the incubation period remains unchanged, and illuminating is performed at fifteen minute intervals.
• B(a)P was assayed at final concentrations of 15.1 and 7.6 ug/ml using Bacillus subtilis strains 11 and TKJ6321 (19).
• B(a)P was assayed at final concentrations of 15.1 and 7.6 ug/ml using Bacillus subtilis strains 11 and TKJ6321 (19).
• the DLS patterns generated by this assay comprise Figures 4a-4d.
• 180 ul each of "EMULPHOR” and “COREXIT” are added to each 5 ml of deionized water and mixed.
• 180 ul of S-9 fraction is added along with appropriate amount of lyphilized bacteria and medium required to achieve a cell concentration of 10 6 bacteria/ml.
• the scoring techniques of the bioassay measure the height of the DLS pattern as a function of bacterial count and the amount of shift in the peaks of the chemical sample pattern and the control sample pattern to determine the degree of morphological change in size and shape of the bacteria.
• the derived indices provide an immediate preliminary indication of the toxic response, the DNA damaging (genotoxic) potential, and identify isosets for compound identification.
• each pattern's height provided an indication of bacterial count and is therefore the principal indicator of toxicity.
• scoring techniques for the identification of morphological changes it was noted that there is a function which is continuous on a closed interval from the beginning of the pattern to its end. Each line segment between any two adjacent points formed an arc, and associated with each point on the X-axis is only one point on the pattern. Using a digitizing tablet, it is possible to obtain a sufficiently large number of points so that the sum of the distances between all adjacent points would approximate the length of the pattern. This was defined as the arc length of the curve. Variances of the arc length correspond with the variances in the bacteria morphology, especially as they related to individual cell size.
• the percent change between the test pattern's arc length and the baseline pattern's arc length was calculated. To determine the fourth order arc index, the arc percent change was divided by 5 and, if the change was positive, 1 was added. If the change was negative, 2 was subtracted. The integer value was retained.
• Each of the three digits, from left to right in the PRI index, derives from the following table according to the fourth order indices of the patterns, starting with the control at "0" time as the reference and the control at 60 minutes as the first digit.
• the tertiary responses for each of the indexed patterns were obtained by multiplying the fourth order Y index by the fourth order arc index.
• the secondary response index (SRI) was determined by the calculation of the sum of the tertiary indices for all indexed patterns combined. This method of calculation is illustrated by the following data derived from an assay:
• the interpretation of the PRI of 132 is that the control of sixty-six (66) minutes displayed normal growth, hence the number 1 as the first digit.
• the treated sample at time "0" showed a loss in bacterial count accompanied by size increase, hence the number 3 as the second digit.
• sixty-six minutes the treated sample showed an increase in bacterial count accompanied by a size increase, hence the number 2 in the third digit.
• PRIs Two extreme examples of PRIs are 133 and 144. These examples are immediately toxic responses from which the cells fail to recover.
• the 133 PRI shows cell enlargement, and the 144 PRI shows cell shrinkage, both being associated with the failure of the cell to recover but by different mechanisms of action.
• the presence of a 3 or 4 in the first digit of any PRI should mean that the assay is invalid because the bacterial control culture is not displaying normal growth.
• a screening score S provides a precise measure of bacterial growth or inhibition of bacteria in the test sample as compared to the control bacteria. It is defined in terms of DLS placement scores and was implemented by previous studies. Wyatt, P.J., Phillips, E.T., Scher, M.G., Kahn, M.R., and Allen, E.H., J. Argic. Food Chem. 25:1080 (1977); Wyatt, P.J., Scher, M.G., and Phillips, D.T., J. Agric. Food Chem. 25:1086 (1977). Thus,
• Dbc is the DLS displacement of the assay broth plus bacteria relative to the assay broth (control)
• Dc is the displacement of the control blank relative to itself (0 when there is no background interference)
• Dbt is displacement of test material in assay broth plus bacteria relative to the control
• Dt is displacement of the test material in assay broth relative to the control blank.
• the untreated "0" time control is represented by a solid line the one hour untreated control by a dot broken line ( . . . . ) , the
• Figures 2a through 2d show the DLS response patterns for strains 6 (rec 34) and 11 (168 wild type) respectively. From the mean Y values of the "0" time and 1 hour samples, it is clear that increases in cell counts occurred in all untreated controls. From the mean Y values of the "0" time and 1 hour samples, it is clear that increases in cell counts occurred in all untreated controls. From the mean Y values of the "0" time and 1 hour samples, it is clear that increases in cell counts occurred in all untreated controls. From
• Figures 3a and 3b show the DLS response patterns for strains 6 (rec E4) and 11 (168 wild type) respectively. From the mean Y values of the "0" time and 1 hour samples, it is clear that increases in cell counts occurred in all untreated controls. From Figure 3a, it is clear that 3.6 ug/ml of MNNG caused a decreased arc length and a curve shift to the left, relative to the light scattering angles of the negative time "0" control. The relative intensity increased less than did the negative control from the "0" to 1 hour interval.
• Strain 11 at the concentration of 3.6 ug/ml had a PRI value of 111, thus showing no compound-related toxicity expressed either immediately or over the 60-minute period following treatment. This is a genotoxic response expressed at the primary response level. Additionally, the SRI value for strains 6 and 11 were -3 and 19, respectively. The negative secondary response of strain 6, when compared to the positive SRI of strain 11, indicates that there is a genotoxic effect which can be repaired by the wild type (strain 11) but not the Rec- mutant (strain 6). Lindane DLS Toxicity Responses:
• Figures 5a through 5d show the DLS response patterns for strains 10 (FH2006-7) and 11 (168 wild type). From the mean Y values of the "0" time and one hour samples, it is clear that increases in cell counts occurred in all untreated controls. From Figures 5a and 5b, it is clear that 107 ug/ml and 53.6 ug/ml of
• Strain 10 at 107 and 5 3.6 ug/ml had PRI values of 222 and 131, respectively. At the 107 ug/ml level, a toxic and a sustained effect (cell enlargement) was seen. However, at the 53.6 ug/ml level, the bacteria recovered after 60 minutes. Strain 11 had PRI values of 131 at both concentrations. This indicated that at both levels, cell lethality and enlargement occurred but that full recovery resulting in growth and the return to normal size occurred by one hour. Although strain 10 may be slightly more affected than strain 11 at the higher concentration of Lindane, the differences in the PRI values do not appear to be useful in making toxicity assessments. The SRI values, however, were substantially different for strains 10 and 11.
• the SRI values for strain 10 at 107 ug/ml and 53.6 ug/ml were -19 and 7, respectively.
• the SRI value was 10 for strain 11 at both 107 ug/ml and 53.6 ug/ml.
• the dose-related SRI trend seen in strain 10 but not in strai 11 indicates a genotoxic effect of this chemical, as demonstrated by the negative SRI value at the higher dose level.
• strain 11 at both concentrations was capable of repairing genetic damage and dividing normally.
• Strain 10 was capable of repairing genetic damage and dividing normally only at the lower concentration of 53.6 ug/ml. From these results, it is clear that primary cytotoxicity may be expressed by a three digit number.

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