Mutation Research, 31 (1975) 365-38o

© Elsevier Scientific Publishing Company, Amsterdam--Printed in The Netherlands

365

T H E QUANTITATIVE MICROSOMAL MUTAGENESIS ASSAY METHOD

C. N. FRANTZ AND H. V. MALLING Environmental Mutagenesis Branch, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 (U.S.A.)

(Received April 4th, 1975) (Accepted May 22rid, 1975)

INTRODUCTION Mammals can convert some non-mutagenic compounds (promutagens) to highly mutagenic metabolites. Such promutagens will not be detected by mutagenicity screening techniques which use microorganisms to detect genetic damage unless mammalian metabolism is first allowed to act on the chemicals. Also, the active mutagen metabolites may be short-lived, such as alkylating agents which combine with many common chemical groups, so that the organism must be in close spatial and temporal contact with the metabolism of the promutagen in order to detect mutagenic activity 1°. The microsomal mutagenicity assay is an in vitro technique which allows tissues to activate promutagens in the presence of a test organism in which induced mutation frequencies are simply determined. Basically, test organisms such as Salmonella typhimurium histidine auxotrophs are incubated at body temperature with mammalian tissue (usually liver microsomes), the promutagen to be tested, and whatever cofactors are necessary for biotransformation of the promutagen to mutagenic metabolites. The specific system described in this paper utilizes fresh mouse or rat liver microsomes in the presence of oxygen, Mg *+ and NADPH (cofactors) to dealkylate dimethylnitrosamine (DMN) and diethylnitrosamine (DEN), producing alkylating agents which revert S. typhimurium His G46. Any microorganism mutation detection system can be used so long as it is not seriously impaired by the requirements of in vitro metabolism. Test organisms which have been used include histidine auxotrophic strains of S. typhimurium, which can detect a wide range of mutagenic activity by means of the various specific reversion mechanisms built into these strainsl,~, s. Bacillus subtilis 168 ilv leucine and valine auxotroph reversion 1', Escherichia coli reversion 15, forward mutations at ad-3A and ad-3B in Neurospora crassa, a funguslL and induction of gene conversion and mitotic recombination in the yeast Saccharomyces cerevisiae~, TM. The range of genetic alterations detectable is limited by the type of mutation system used. Therefore, insofar as microorganisms cannot detect all types of genetic damage, e.g., chromosome translocations, a negative result in a microsomal assay does not rule out mutagenic activity of the compound in mammals. The microsomal mutagenesis assay has not yet be~n successfully performed with cultured mammalian cells.

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Any tissue preparation can theoretically he used to activate a promutagen. However, the vast majority of compounds that should and will he tested for metabolically activated mutagenicity are foreign compounds not closely resembling those normally found in manmlals. Tile microsomal fraction is where such compounds would most likely be metabolized. Microsomes are derived from endoplasmic reticulum, are found in the 9ooo g supernatant of mammalian tissue homogenates, and can be prepared in pure, active form by centrifugation of the 9ooo g supernatant at I o5,ooo g (or at I5oo g in the presence of calcimn). Liver is the tissue richest by far in the microsomal enzyme activity, yet there are marked age, species, sex and strain differences. Also, some reactions which b a r d \ ' proceed in the liver m a v occur readily in lung, kidney, or gut epithelium. The major microsomal enzyme reaction mechanisms, known as "mixed function oxidases", typically involve enzymatic hydroxylation ; for example, DMN demethylation probably occurs when one methyl group is oxidized, forming a highly unstable hydroxylated compound, which dissociates into formaldehyde and monomethylnitrosamine, which in turn is highly unstable and spontaneously degrades to release a nmthonium ion which will methylate m a n y common groups ~. Many commonly used drugs are metabolized by this system. The essential make-up of this membrane bound enzyme complex is not completely characterized, but one comFonent enzyme of all the reactions which can be easily measured is cytochrome P-45o, the terminal electron acceptor of the system. Energy for the reaction is generally provided by N A D H or NADPH, which are therelore necessary (and labile) cofactors and which must be provided for in vitro reactions. Other cofactors, such as calcium or magnesium ions, vary with the specitic enzyme activity studied. Some types of metabolic activation are not readily reproducible in vilro. For example, activation of the highly carcinogenic compounds 2-acetylaminofluorene, and subsequently of its microsomal metabolic product, N-hydroxyacetylaminofluorene, probably takes place in vitro and to some extent in vivo by a transacetylation reaction, and thus is nmtagenic in the basic microsomal test system 1. However, by far the most potent metabolic product in terms of binding to DNA is the sulfate ester, which can only be found in the presence of sulfotransferases (found in the cytosol) and the coenzyme adenosine-5'-phosphosulfate (PAPS) or a similar source of high energy sulfate ~a. If little or nothing is known of the metabolic activation of the pronmtagen to be tested, negative results are likely to occur due to lack of provision of proper cofactors (diverse metal ions, various phosphorylated nucleotides, or soluhle enzymes). Some reactions which can be produced i,a vitro may not occur to any significant extent i,~ vivo. For example, nitrofurans, metabolized in the presence of oxygen by some oxidases yield different quantities of mutagenic products than when metabolized anaerobically (by different oxidases) '~,n,t~. Some mixed function oxidase reactions can be performed without enzymes using ascorbic acid 9. Microsomes also perform reductions, hydrolyses and conjugations when appropriate cofactors are available. Many of the same type of reactions occur in the cytosol, even in mitochondria, but usually are specific for substrates occurring naturally in mainmalian tissue. In summary, use of microsomes and NADH and N A D P H to screen in vitro for mutagenic metabolites when the specific biochemistry is unknown is a very limited

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screening test because (a) only mixed-function-oxidase reactions will be seen in quantity; (b) some mixed-function-oxidase reactions will not be seen since other needed cofactors (such as metal ions) will not be present in sufficient amounts, (c) although conjugation reactions generally detoxify compounds and allow excretion s, conjugations (glucuronide formation, methylation, acetylation, etc.), reductions, and hydrolyses will probably not be seen because proper cofactors will not be present in significant quantity. Finally, some reactions which do not occur significantly in vivo may be seen in vitro. Thus, although an intraperitoneal host-mediated assay may be much less sensitive, it is more appropriate for screening compounds the possible metabolism of which is unknown. Furthermore, the intrahepatic host-mediated assay is the most sensitive mutation detection technique yet devised employing relatively inexpensive mammalian metabolism and microorganisms1°, 14. The microsomal mutagenesis assay is appropriate as a screening technique only where a specific type of metabolic activation to a mutagenic product is highly suspected. However, it is a very useful technique for determining the character and kinetics of the biochemistry involved in activation or deactivation. Essentially, a biochemical enzyme assay is performed .The reaction product is measured as mutagen activity. To determine the exact activation pathway, both mutagenicity and biochemical product must be measured. SPECIAL APPARATUS

The microsomal mutagenesis assay may be divided into five essential parts, (a) preparation of microsomes, (b) preparation of microorganisms, (c) the actual incubation of microorganisms, microsomes, and cofactors, (d) plating and incubation of test organisms, and (e) counting of test organisms.

Preparation of microsomes Tissue homogenizer of the Potter-Elvehjem type in which a teflon pestle fits snugly into a glass tube containing tissue and solution. An elaborate foot pedal operated homogenizer kit with tubes is available from Tri-R-Instruments (Stock No. S-2oo). Alternatively, a grounded heavy duty I/4" electric hand drill may be obtained at a local hardware store and two pestles (24.54 mm diameter), (Stock No. S-23) and four tubes to fit (Stock No. S-37) (tube capacity about 4 ° ml) are adequate. Also, many find the hand drill an easier way to homogenize in ice to keep the preparation cooled. A high speed refrigerated centrifuge is needed for the first ce~ltrifugation of the tissue homogenate at between 9000 g and 20,000 g. Models adequate for the job start at around $3,500.00. Alternatively, if a good quality refrigerated centrifuge is already available, converter kits for high speed angled rotor operation are available. Extra equipment. If biochemical quality work is considered, purification of microsomes is required in order to get a reliable measurement of microsomal protein for standardization. Purified microsomes also may be less damaging to delicate test organisms than a simple liver homogenate 9000 g supernatant. Purification may be performed on a preparative ultra-centrifuge at lO5,OOOg. Alternatively, the calcium precipitation technique allows shorter centrifugation time at 15oo g, so only a higher capacity head for a high speed (up to 9000 g) refrigerated centrifuge would be needed.

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Incubation of reaction mixtures Mechanical timer and water bath shaker. Microsomal enzyme reactions generally proceed optimally at around 37 ° in the presence of oxygen. If the reaction is performed in unstoppered flasks on a water bath shaker, exact temperature control is maintained and sufficient oxygen reaches the microsomes during shaking. A water bath shaker with a rack holding 9 25o-ml erlenmayer flasks is adequate. However, if there is a chance that active metabolites are volatile, flasks should be stoppered sterilely after gassing with pure oxygen. A small oxygen tank, holder, and regulator with gauge is then needed. Stoppering will also prevent accidental contamination during incubation. Ideally, reactions producing volatile carcinogens or mutagens should be done inside an exhaust hood which filters exhausted air. Extra equipment. If biochemical reactions are to be monitored precisely, a footpedal on/off switch for the shaker-incubator and a stop watch are desirable. Plating and incubation of test organisms With S. typhimurium, test organisms are most easily plated in agar overlays which can be conveniently melted either b y autoclave or by boiling water bath. However, a small autoclave is necessary for preparation of sterile solutions. 45 ° water bath. Bacterial plating overlays must be kept melted until ready for use. Test organism colony counting Extra equipment. A single button hand-held tabulator aids in counting colonies. More elaborate devices include counters that operate when the plate is touched or automatic computerized colony counters (about $5o00 from Fisher Scientific). Other equipment. Precision balance to weigh out NADPH, media components, mutagens, etc. Trip balance to balance centrifuge tubes. A p H meter is convenient for adjusting p H of stock solutions. A small refrigerator is necessary for storage of media, solutions, and bacteria on agar slants. Glassware and routine nonchemical supplies for preparation of media and solutions 6 IOOO-ml erlenmeyer flasks; I i 25o-ml erlenmeyer flasks; 2 I25-mi screw top bottles; io I25-ml erlenmeyer flasks; 20 14 × 12o m m screw cap tubes. Glassware and routine nonchemical supplies neededfor a 9 sample microsomal mutagenesis assay with S. typhimurium histidine auxotroph Reusable. 3 I25-ml erlenmayer flasks with stoppers; IO 25o-ml erlenmeyer flasks with stoppers; I 5oo-ml erlenmeyer flask with stopper; 4 ° 20 × I5o-mm test tubes with caps; 60 13 × i o o - m m test tubes with caps; 2 5o-ml capped centrifuge tubes ; 4 5o-ml beakers; 2 5o-ml capped high speed centrifuge tubes ; I small ice bucket ; I large ice bucket or tray; I hand pipettor; 12 Io-ml pipettes; 18 o.i-ml pipettes; I autoclavable fiberglass air filter; 2 large surgical scissors; 2 blunt surgical forceps; I fine surgical forceps; I 25o-ml beaker; I 9-inch section of wooden gutter; 2 push pins; I plastic squeeze bottle (200 ml). Disposable. 60 IOO × 15 m m plastic petri dishes ; 3 mice. Chemical supplies for media and solutions prepared before experimentation Distilled water; MgSO4 '7H20 1og ; citric acid I oog; KH2PO45oog; NaHNH4HPO~-

MICROSOMAL M U T A G E N I C I T Y A S S A Y

369

4H20 175 g; agar (Bacto) 22 g; glucose IO g; 1-histidine 21 g; biotin 12.2 mg; NaC1 18.5 g; Difco-Bacto nutrient agar nutrient broth dehydrate; MgCI~.6H20 61o mg; Tris-HC1 (Sigma 7-9) 3.03 g; I N HC1 20 ml; sucrose 21.2 g.

Chemicals for 9 sample assay T P N H (NADPH) 4 ° mg (alternatively, a T P N H generating system may be used which is less expensive1*); DPN (NADH) IO mg; mutagen. Costing Once the technique has been established in a given laboratory, a single technician, efficient but not necessarily with an extensive background, should be able to prepare microsomes and run 36 samples at 2o-min incubation each, dilute, and plate the samples in an 8-h day. Hand counting the plates should take about 4 h, while preparation of media for this many tests should take about 4 man h. Thus, one experimental point could be determined on an average every 1/2 man h. PRELIMINARY PREPARATION

Test organism Many scientists now have the S. typhimurium histidine auxotroph tester strains developed by BRUCE AMES, University of California, Berkeley. Tester strains should be chosen carefully with the possible mechanism of mutagenic action in mind*. Grow up overnight at 37 °. Dip a sterile stick into the broth and streak onto a number of nutrient agar slants. After 24 h of 37 ° incubation, bacteria should be visible on slant. Use these slants to inoculate, with a sterile stick, nutrient broth flasks for each experiment. To check for contamination and spontaneous reversions of your stock bacteria, simply grow up bacteria at appropriate concentrations on minimal media plates in overlays with and without 1-histidine and check colony morphology. Solutions for microsome preparation and buffer Solution A. MgC1, '6I-I20 o.61 g; Tris (Sigma 7-9) 3.03 g. Dissolve in approx. 60 ml water. Adjust to p H 7.5 by adding approx. 20 ml I N HC1 then fill up to IOO ml. Adjust p H after autoclaving. Store in refrigerator. Solution B (0.62 M sucrose). Sucrose 21.2 g. Add water until approx. 7 ° ml in a volumetric flask. Can be autoclaved. After autoclaving, add sterile distilled water to IOO ml. Store in refrigerator. Media and stock solutions for Salmonella Earl's salts stock solution ( 5 0 × ) . Distilled 1-I20 670 ml; MgSO4.TH,O IO g; citric acid IOO g; KH, PO, 500 g; NaHNH, HPO, .4I-I20 175 g. Important: add each chemical separately and in order. Dissolve each one before adding the next. No more than 5 ° g of KH, PO, should be added at one time. After chemicals are dissolved, pour approx. IOO ml in each 25o-ml flask, then autoclave for 15 min. Plating medium. Distilled I-I20 500 ml; agar 8.0 g (agar must be washed three times before use, as unwashed agar may be contaminated with histidine) ; glucose IO g. Wash agar 3 times; add io g glucose. Add distilled H,O up to 5oo-ml mark.

37 °

C. N. F R A N T Z

A N D H . V. M A L L I N G

Place magnetic stir bar in the flask. Autoclave for 15 rain (sterilized). It is best not to make up smaller batches as washing m a y deplete agar concentration. Immediately after autoclaving, add the following to the flask. salt solution (5ox) (No. I) IO ml; histidine stock solution (No. 3) 1.25 1111; biotin stock solution (No. 4) I.o nil. Shake the medium gently. Keep in 45 ° water bath for I h. Pour apt)rox. 20 ml in each IOO × 15 mnl petri dish. Refrigerate petri dishes. l-histidine stock solution. Add 21 mg 1-histidine to IOO ml H,,O (use screw top bottle). Autoclave for 15 rain. Keep in refrigerator. Biotin stock solutions. Add 12.2 lng biotin to IOO lnl H20 (use screw top bottle). Autoclave for 15 min. Keep in refrigerator. l-histidine soft agar. Distilled H20 IOOO ml; NaC1 5 g; 1-histidine 39 rag; agar (Bacto) 7 g (agar should be washed three times). Melt agar. Dispense 2 ml in each 13 × IOO ram-test tube. Plug with metal cap. Autoclave for IO min. Store in refrigerator. Soft agar minus histidine. Distilled H20 I 0 0 0 I n l ; NaC15 g; agar (Bacto) 7 g (Agar should be washed three times) Melt agar. Dispense 2 ml in each 13 × Ioo-mm test tube. Plug with metal cap. Autoclave for io min. Store in refrigerator. Salmonella growth flasks. Nutrient broth: follow the directions on bottle. Use Difco-Bacto nutrient broth; dehydrate. Add 5 ° nil of the medium to 125 ml flask. Seal with a lunfinum foil. Autoclave for 15 rain. Refrigerate. Saline. Add 8.5 g NaC1 in IOOOml H~O. Dispense approx. 15o ml in 25o-ml flask. Cover with metal cap. Autoclave for 15 min. Refrigerate. Nutrient agar slants. Follow directions for nutrient agar. Place in screw-cap tubes. Autoclave. Slant (approx. 45°). Refrigerate. Sterile distilled u,ater. Autoclave in 5oo ml aliquots. Refrigerate. Autoclave glassware, pipets, stoppers, oxygen filter, etc. Bacterial concentratio~z It is preferable to consistently treat the same number of bacteria. Wtlen the bacteria are grown up overnight, they are in a non-log growth phase, as m a n y nutrients are used up. Usually about the same number will be consistently grown up. However, in order to determine more exactly the number of bacteria treated, the logarithmic relationship of optical density to bacterial concentration should be determined separately for each test organism (and spectrophotometer) used. A graph of this relationship should be constructed by resuspending the centrifuged pellet of S. typhimurium grown up overnight in about 7 ml of iced sterile saline and making small step-wise dilutions. Determine optical density by adding o.25 ml of each dilution to a separate spectrophotometer cuvette containing 4.75 ml iced saline. Read O.D. at 42o nm. To determine the number of bacteria, make a further dilution b y a factor of 5" lO4 in iced sterile saline of any one of the above original dilutions, plate in triplicate o.5 or i.o ml in histidine-containing overlay, incubate 48 h, and count the colonies. Calculate the number of bacteria in the original dilutions and plot against O.D. (The graph will be accurate in the range of 6o-8o% O.D. Always read O.D. at the same sample temperature.)

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STEP-BY-STEP

The microsomal mutagenesis assay (9 samples) using liver homogenate supernatant and S. typhimurium His G46. (I) Inoculate His G46 S. typh~murium bacteria from agar slant into 5o ml nutrient broth the day before experiment is planned, grow up overnight (16-18 h) at 37 °. Bacteria will be in stationary growth phase. (2) Take NADPH (TPNH), other cofactors, mutagens, etc. from freezer to warm to room temperature before opening containers. (TPNH should be stored in freezer in desiccant.) (3) Make up Solution C on ice by adding 20 ml Solution A, 20 ml Solution B, IO ml cold sterile H20. Mix. Keep on ice. (4) Make up a set of 0.85% saline dilution tubes (capped, 20 × 15o nm) on ice for each of the nine mutagenesis flasks as follows: 9.5 ml, 9.9 ml, 9.9 ml, 4 ml. (5) Refrigerate or place on ice all solutions and equipment. Prewarm petri dishes containing minimal medium in incubator. (6) Centrifuge bacterial broth at 2000 rpm for 20-35 min at 4 °. Decant supernate, and resuspend bacteria pellet in lO-2O ml (see above) of cold normal saline by gentle shaking. Do not vortex. Keep on ice until use. (7) Microsome preparation. (a) Kill 3 animals. Allow to bleed by decapitation in order to have as little blood in livers as possible. Pooling livers from three animals will reduce individual variation in enzyme activity. (b) Remove livers using sterile technique. (With mice, it is perhaps easiest after bleeding to first place the mouse on its back on a 9" section of wooden gutter, rinse the abdomen with alcohol from a squeeze bottle, push pin its tail to the board, and remove skin from abdomen (not necessary to use sterile instruments). Strip the skin over the mouse's head and push pin it to the wooden gutter. Rinse abdominal wall with alcohol. Using sterile forceps and scissors (kept in alcohol in a 25o-ml beaker), open abdominal wall. It is best to remove the gall bladder, which in poorly kept mice may be infected with E. toll, and probably is infected with anaerobes. Using sterile fine scissors and forceps (kept in sterile H~O or saline in a 50 ml beaker) grasp liver, pull and cut away fine attachments to diaphragm and cut hepatic veins. Pull liver away from animal and cut attachment at ligament of Treitz. Place liver in 50 ml sterile beaker containing IO ml Solution C. Alternatively, cut portions from each lobe of the liver avoiding the gall bladder, and do not try to remove the entire liver. (c) Rinse twice in ice cold Solution C (enough to cover livers) in 5o-ml beakers by transferring livers. (d) Weigh livers in tared 5 ° m l beaker containing a known amount of iced Solution C and adiust volume as desired. (4-5 ml/g liver is standard, while 2 ml/g liver is quite concentrated.) (e) Homogenize on ice in a precooled glass tube with cold teflon pistil (tight-fitting) ; two or three passes through suspension is sufficient. (f) Rinse centrifuge tubes with alcohol to sterilize, and rinse with iced sterile distilled water or saline. Keep on ice.

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(g) Centrifuge at 20,000 g for IO min at 4 °, or at 9000 g for lO-2O min. (h) Decant completely the supernatant into a cooled container. Mix well before each use. (8) To force DMN demethylation, 0.8 mg T P N H / m l of reaction mixture or even more is necessary. However, m a n y biochemical reactions proceed at m a x i m u m rates with less than IOO /,g/ml of T P N H . Weigh out T P N H and mutagen. Do not dissolve T P N H or D P N H until just prior to use. Note. It is simpler to prepare all solutions to be added to mutagenesis flasks so that I ml of each is added. Thus a stock solution of m u t a g e n would be five times the desired concentration, as five items (i ml each) are added to each flask. (9) Place a 25o ml sterile flask on ice for each sample to be run. A d d i ml each of Solution C, liver homogenate supernate, and bacteria to flasks. Add m u t a g e n unless is extremely short-lived. Dissolve T P N H in iced sterile distilled water. To start reaction place flasks on shaker water b a t h at 37 ° for I min (to w a r m up) then add I ml T P N H to each at timed intervals. Through a sterile filter, gas each flask 1-2 sec; stopper. Time reaction from addition of T P N H . After 15-3o min microsomal reactions generally become non-linear with respect to time. (io) To stop reaction remove 0. 5 (or i.o) ml aliquot from flask, place in 9.5 (or 9.o) ml first saline dilution tube (iced). (ii) Dilute for viable counts, mixing well at each step. o.5 flask aliquot

- ~

9.5 first dilution tube

0.I ml - "

9.9 ml

O.I inl -"

9.9 ml

I.O ml --

4.0 ml last dilution tube

(12) Melt agar overlays b y autoclave or boiling water, keep at 45 °. (13) Plating. (a) A d d o. 5 ml of last dilution tube to histidine-containing overlay, mix, spread evenly on plate. Repeat once for duplicate plates of viable count for each sample. (b) A d d o. 5 ml or i.o ml (depending on expected n u m b e r of revertants) of first saline dilution tube to histidine-free overlay, mix, spread evenly on plate. Repeat 3 times for quadruplicate plates of the revertant count for each sample. Swirl overlay around on plate while still molten to distribute bacteria evenly for easier counting. (14) I n c u b a t e plates at 37 ° . Turn plate over after overlay has hardened to avoid condensation dripping onto plates. After 36 h colonies are visible. B y 48 h t h e y are easily counted. (15) Count colonies. For a balance between ease of counting and significant, reliable results, aim for about 2oo colonies per plate. Resuspension in 15 ml saline of the bacteria grown to stationary phase in 5o ml nutrient b r o t h should give about 4oo organisms/ml in the last dilution tube if there is no significant killing. If contamination is seen, it is best to discard the experiment. An easy w a y to demonstrate t h a t Salmonella are what you have recovered (personally tried so far only with G46 and TAI535) is to demonstrate t h a t the bacteria agglutinate on a slide in the presence of Salmonella group B antisera. This technique does not rule out contamination with other group B Salmonella.

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(16) Calculations. The possibility of differential survival between revertants and auxotrophs is never ruled out absolutely without a reconstruction experiment for each mutagen; it is best to calculate % survival and to base calculations on the number of revertants per surviving bacteria only with that possibility in mind, and to ideally perform a reconstruction experiment for each mutagen tested. When the ratio between induced and spontaneous mutants is low and test organism survival is low, a reconstruction experiment demonstrating equal survival of auxotrophs and revertants is mandatory. (a) surviving organisms: survivors/ml first dilution tube histidine requiring colony count total × dilution factor (ml of last dilution tube/plate) (number of plates) (dilution factor is 5" lO4 in above instructions, i.e. the dilutions between the tube from which revertants and the tube from which survivors are determined.). (b) % survival =

experimental survivors/ml ist dilution tube control survivors/ml Ist dilution tube

(c) revertants : revertants/ml Ist dilution tube

histidine non-requiring colony count total (ml of first dilution tube/plate) (number of plates)

(c) revertants/ml first dilution tube (d) revertants/Io ~ survivors= - - -(a) -- lO s survivors/ml first dilution tube (17) The basic statistical approach is a chi square comparing number of bacteria and number of revertants in experimental and control flasks. APPENDIX A

Preparation of Ca 2+ precipitated microsomes ~ Refer to 9ooo g liver homogenate supernatant technique for completeness. (I) (a) Make up solutions for liver preparation (sterile) MCI, MC2, etc. denote different solutions. The composition of these solutions are given at the end of the appendix. Homogenizing solution, I part MCI : I part MC2; Diluting solution, I part MC2 : 19 parts H~O: 20 parts MC3; Sucrose solution, I part MC2:19 parts H~O; KC1 resuspension = MC4; Keep on ice or at 4 °. (b) Using sterile technique, remove livers, place immediately in iced Homogenizing Solution, rinse 2 times in Homogenizing Solution. Weigh livers. Make up to 4 ml Homogenizing Solution/g liver. Homogenize on ice. (c) Spin at 4 °, IO,OOOg for 20 min (ss34 Sorval 9500 rpm). Pour off supernatant. Discard pellet. (d) Dilute supernatant i : 6 (by volume) with Diluting Solution in large centrifuge tube. Spin at 15oo g for lO-2O min at 4 °. Decant supernatant. (e) If washed microsomes are desired, as for cytochrome P-45o determination, loosen pellet in a few ml of Sucrose Solution by vortex. Place mixture in sterile cold homogenizing tube and homogenize by hand on ice. Rinse back into large centrifuge tube with cold Sucrose Solution and make up to equal previous volume. Spin at 4 °, I5oo g

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for IO 20 min. Decant supernatant by suction through sterile pipet, discard. Loosen pellet by vortexing in a few ml of 1.15% KC1 (MC4). Re-homogenize by hand in sterile cold homogenizing tube with teflon pistil. Rinse into sterile, cold, capped graduated cylinder. If a high concentration of enzyme is desired, use very little KC1. (2) Protein determination (LowRY) 7. Make up stock solutions prior to experiment. (a) Add 2.o ml H20 to each 18 × I75 mm test tube needed. (b) Standards, add o, 5o, ioo, 15o, 2oo, and 25o pl of bovine serum albumin (I nlg/ml) to each of 6 test tubes in duplicate. (c) Add 5, IO, 15, 2o/~1 of well mixed sample (or of I. I eaO/D C~..o KC1 suspension for control) to 4 respective test tubes. (d) Add 2.5 ml CuSO4 solution and 2.5 ml tartrate solution to 25 ° m l graduated cylinder, mix welh Then fill up to 25 o m l with Lowry A Solution. Add IO ml to each tube. Mix. (This solution must be made just prior to use.) (e) Wait IO min. (f) Dilute Folin-phenol reagent I : i with distilled H20 ; add I nfi to each tube. Mix. (g) Wait 2o rain. (h) Mix. Read O.D. at 66o nm. (i) Construct graph of standards. (j) Substract buffer reading from each sample, read protein concentration from net O.D. of sample on straight line portion of graph. Average results from the different concentrations. (3) Adjust microsomal protein to desired concentration with cold I.I5°:~) KC1.

Microsomal preparation solutions M C I Tris-HC1 1.21 g; MgC12-6H20 o.2o 4 g; KC1 o.371 g; CaCI2 o.I78 g. Dissolve the above in 0o ml of distilled water. Autoclave. Adjust pH to 7.5 with I N HC1. Make up to ioo ml with sterile distilled water. MC2 Sucrose 17.I g. Dissolve in about 7o 1111 distilled water. Autoclave. Add sterile distilled water to IOO nil. MC3 MgCI,~-6H20 0.408 g; CaC12 o.356 g. Dissolve in 15o ml distilled water in a 2oo-ml volumetric flask. Autoclave. Make up to 200 ml with sterile distilled water. MC 4 KC1 1.15 g. Dissolve in 9 ° m l distilled water. Autoclave. Make up to ioo ml with sterile distilled water. Reagents for Lowry determination 2% CuSO~. 5H20 (stock solution)" 4°//o K Na tartrate (stock solution). Lowry "A"" NaOH 8 g; Na2COa 6o g. Dissolve in 2000 ml distilled water (stock solution). Folin-Cocteau Phenol reagent (commercial) ; Bovine serum albumin I mg/ml (stock solution). Refrigerate. APPENDIX

B

Factors affecting the microsomal activation of D M N and D E N (I) Reaction mixture components. Microsomes, NADPH, and oxygen are absolute

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TABLE I EFFECTS OF TREATMENTCONDITIONSON THE FREQUENCYOF HISTIDINEREVERSION INDUCED IN S. typhimurium STRAINTAI53O Treatment

% Survival

Number of revertants

Revertants per lO s survivors

Complete system Complete system plus N2 minus O3 Minus NADPH Minus microsomal fraction Minus MgC13 Minus DMN (control)

i 14 95 121 i26 lO9 ioo

916 2 I o 20 o

8.16 o.o2 o.oi o o.19 o

2o,ooo g supernatant of male B6CDFI mouse livers prepared at 5 ml buffer/g liver was incubated 20 rain with 3.2 mg NADPH and 45 mM DMN, and gassed with either lOO% N3 or lOO% 03. r e q u i r e m e n t s for the i n d u c t i o n of m u t a t i o n s with DMN (Table I). W h e n a n y one of these c o m p o n e n t s were deleted from the reaction mixture, no reversions were obtained. W h e n the ionic cofactor m a g n e s i u m was left out of the reaction mixture, significant m u t a g e n i c a c t i v i t y was obtained, b u t almost two orders of m a g n i t u d e less t h a n with MgC1, (see ref. 8). The a c t i v a t i o n of DMN without added MgC12 m a y be due to Mg 2+ already present in the liver homogenate s u p e r n a t a n t . (2) Comparison of buffers. The a c t i v a t i o n of DMN to a m u t a g e n was compared in two different buffers (Table II). No significant difference in survival or reversion frequency with DMN was found between H E P E S a n d Tris buffers, both at p H 7.4. TABLE II COMPARISON OF THE EFFECT OF T R I S AND H E P E S BUFFERS ON THE FREQUENCY OF HISTIDINE REVERSION INDUCED IN S. typhimurium STRAIN T A I 5 3 0 INCUBATED WITH MICROSOMES

Buffer

DMN

Percent Survival

Revertants counted

Revertants/per zo ~ survivors

Tris-HCl 200/*moles Tris-HCl 200/,moles HEPES 200/*moles HEPES 200/*moles

9o mM 90 mM

ioo 87 84 81

3 1168 3 1389

o.03 i 1.6 o.o3 14.8

The microsomes were prepared as the 9000 g supernatant of B6CDFI male mouse livers homogeni z e d in 5 m l 0 . 2 5 M s u c r o s e p e r g r a m l i v e r . T h e 5 m l m i x t u r e , i n c u b a t e d i n a 2 5 o - m l f l a s k f o r 2 0 miD, c o n t a i n e d 2 o 0 / , m o l e s T r i s - H C 1 o r H E P E S b u f f e r p H 7.4, 2 4 / , m o l e s MgC1 v 1 . 8 / * m o l e s N A D P H , 5o0/*moles sucrose and i ml of mouse liver homogenate 20,00o g supernatant.

(3) D u r a t i o n of incubation. The reaction m i x t u r e was i n c u b a t e d at 37 ° for v a r y i n g lengths of time in the presence of DMN (Table III). Although the bacteria were in s t a t i o n a r y growth phase at the start of the experiment, i n c u b a t i o n clearly allowed t h e m to multiply. I n d u c t i o n of reversions per IO e s u r v i v i n g bacteria increased in a linear fashion for 30 miD, b u t h a d decreased d r a m a t i c a l l y when m e a s u r e d after 60 m i n of i n c u b a t i o n . This finding suggests a difference between r e v e r t a n t s a n d histidine requiring cells in a b i l i t y to survive or m u l t i p l y in a 6u-miD incubation. (4) The frequency of reversions increased with increasing concentrations of DMN in the reaction mixture. E v e n at the lowest concentration, 5.6 m M DMN, there was a b o u t a 6u-fold increase in m u t a n t frequency over control (Table IV). (5) S K F 525-A i n h i b i t i o n of DMN m u t a g e n i c i t y . S K F 525-A was added to the incubation m i x t u r e in v a r y i n g concentrations (Table V). At a high concentration, a signifi-

376

C . N . FRANTZ AND H. V. MALLING

TABLE III EFFECT OF DURATION OF TREATMENT ON MUTAGENICITY OF DMN Duration in min

DMN concentration

°/o B a c t e r i a l survival

Number of revertants counted i n 2.0 m l

Revertants/ 2 o ~ survivors

o 5 io 20 3° 60 60

i8o 18o 18o 18o 18o 18o o

ioo lO6 i 13 166 136 217 2o 4

2 42 365 54 ° 659 761 i

O.lO 2.0 17 34 5° 35 0.o 3

irtM mM mM mM mM mM

A 20,000 g s u p e r n a t a n t of B 6 D 2 F I male m o u s e livers homogenized in 4 ml buffer per g r a m liver a n d a final c o n c e n t r a t i o n of 0.8 n l g / m l T P N H were used. T A B L E IV EFFECT OF DMN CONCENTRATION ON MUTANT FREQUENCY DMN concentration (raM)

O//o B a c t e r i a l survival

Number of revertants counted i n o.25 m l

Revertants / lO s survivors

O 5.6 11. 3 22.5 45

IOO 87.5 85.8 87.5 89.3

1 6O 152 I89 432

O,O2 1.27 3.28 4-73 9.oo

A 20,000 g s u p e r n a t a n t of C3H × C57B/6 male m o u s e livers homogenized in 5 ml buffer per grant liver and a final c o n c e n t r a t i o n of 0.8 m g / m l T P N H were used. TABLE V S K F 525-A INHIBITION OF DMN MUTAGENICITY IN THE MICROSOMAL ASSAY S K F 525-A concentration

DMN concentration

o io m M o IO ml~/ I mM o.i m M

o o 18o 18o 18o 18o

°1o B a c t e r i a l survival

ioo 65 ioo 39 >ioo >1oo

mM mM mM mM

Number of revertants counted i n Lo ml

Revertants/ I o 6 survivors

i.o 1. 3 664 16 1.5 24

0.03 o.II 38 2. 3 0.07 1.2

The 20,000 g s u p e r n a t a n t of B 6 D 2 F I male m o u s e livers homogenized in 4 ml buffered sucrose per g r a m liver was i n c u b a t e d w i t h 18o m M D M N and 0.8 m g / m l T P N H for 2o min. cant percentage quently

of b a c t e r i a

w e r e killed. A t I m M

SKF

525-A, a concentration

found to inhibit mixed function oxidase reactions, no DMN

fre-

induced rever-

sions w e r e o b s e r v e d a n d n o effect o n b a c t e r i a l s u r v i v a l w a s seen. (6) M o u s e s t r a i n d i f f e r e n c e s i n t h e a c t i v a t i o n homogenate incubated

supernatant

was prepared

with DMN or DEN

of D M N

and DEN

to mutagens.

Liver

f r o m t h r e e d i f f e r e n t s t r a i n s of m i c e a n d w e r e

(Table VI). Significant differences were seen between

t h e s t r a i n s i n r e v e r s i o n s p e r lO s s u r v i v i n g b a c t e r i a w h e n i n c u b a t e d w i t h D M N , b u t n o

377

MICROSOMAL MUTAGENICITY ASSAY TABLE VI

STRAIN DIFFERENCES IN THE METABOLISM BY MOUSE LIVER OF DMN AND D E N TO MUTAGENS

Mouse strain

Mutagenic activity---revertants/Io* survivors Control 2oo m M D E N 9° m M D M N

C3 H (Oak Ridge) B6C3FI DBA/2J

0.02 (lOO%) --

o . 5 1 (29%)

--

0.56 (29%)

0.60 (36%) a

41 (93%) 15 (89%) I o (97%)

2o,ooo g s u p e r n a t a n t of i3-week-old male m o u s e livers homogenized in 4 ml buffer/g liver and i n c u b a t e d w i t h 0.8 m g / m l T P N H for 20 min. aFigures in p a r e n t h e s e s are the percentage of bacteria surviving t r e a t m e n t .

differences were seen when incubated with DEN. The differences in liver metabolic activity seen are therefore probably specific for the site of DMN metabolism, and not an effect of microsomalprotein or cytochrome P-45o content per gramof liver. Alternatively, D E N activation m a y be limited in this incubation by the absence of some unknown cofactor such that quantitative data in this particular incubation system are not meaningful. (7) Mouse and rat livers were compared for their ability to metabolize DMN and D E N to mutagens. Dramatic differences between the two species were found for DMN, but none for D E N (Table VII). When microsomes were prepared to the same protein concentration b y the calcium precipitation technique, differences between mouse and rat were much smaller (C. N. FRANTZ AND H. V. MALLING,unpublished). TABLE VII SPECIES DIFFERENCES IN THE METABOLISM BY LIVER OF D M N AND D E N TO MUTAGENS

Species

Compound

Concentration % Bacterial (raM) survival

Number of revertants counted

Revertants[ IO e survivors

Mouse Rat Mouse Rat

Control DMN DMN DEN DEN

o 45 45 45 45

i 967 3° 7 13

o.oi 20.3 0.40 o.14 0.22

ioo 93 87 ioo 7°

2o,ooog s u p e r n a t a n t s of male 12-week-old S p r a g u e - D a w l e y r a n d o m bred r a t livers a n d C3H × C57B / 6 male mouse livers homogenized in 5 ml buffer per g r a m liver and a final concentration of 0.64 m g / m l T P N H and o.56 m g / m l D P N H were used.

(8) Microsomal protein concentration. In two separate experiments, microsomes were prepared and washed, and incubated with DMN and T P N H (Table VIII). Induction of reversions of S. typhimurium His G46 increased dramatically as amount of enzyme in the incubation mixture was increased. (9) Effect of 3-methylcholanthrene pretreatment. Mice of two different strains were treated with 3-methylcholanthrene, and 24 h later microsomes were prepared from the livers of treated mice and matched controls. The microsomes from all four groups were washed and adjusted to identical protein concentrations, and incubated with DMN and T P N H (Table IX). Microsomes from C3H mice activated DMN to a mutagen more than did those of C57BL6 mice. Pretreatment with 3-methylcholanthrene, which is known to induce activity of mixed function oxidase microsomal enzymes,

378

C . N . FRANTZ AND H. V. MALLING

T A B L E VIII T H E E F F E C T O F M I C R O S O M A L P R O T E I N C O N C E N T R A T I O N ON T H E M E T A B O L I S M O F l ) ~ ' I N TO A M U T A G E N

mg microsomal protein /ml in incubation

revertants per r o ~ survivors

1.6

4 l

1.2

r7

~.o 0.8 0.6 o

9 2. 7 {).68 o.o 4

0.75 0.60 0.45 0.30

~.61 0.62 o.42 o.34

O,I 5

0.28

O

O,IO

The upper experiment was done with microsomes prepared in the traditional way, homogenized in 4 ml H E P E S buffer/g B6D2FI male mouse liver centrifuged at io,ooo g, the s u p e r n a t a n t of which was centrifuged at Io5,ooo g twice for i h. i8o m M DMN and o.8 mg/nfl T P N H were used. The lower experiment was performed with C57BL/6 mouse liver microsomes prepared by calcium precipitation and treated with ioo m M DMN and L7 m g / m l T P N H . The results nevertheless correspond closely.

T A B L E 1X EFFECT

OF 3 - M E T H Y L C H O L A N T H R E N E

IN TERMS LIVER

OF MICROSOMAL PROTEIN

MICROSOMES OF

Mouse strain

Inducer

DMN n~g liver

ENZYME

INDUCTION

DMN

YIELD,

ON L I V E R S O F

DEMETHYLASE

Control 3-MC Control 3 -MC

4.6 3.9 4.0 4 .6

AND ('57[~L/0

MICI,;

AND METABOLISM

BY

TO A M U T A G E N

mg mg micro- Demepurified somes per thylase a microg liver activity

% Sur v i v al

sofnes

C57BL/6 C57BL/6 C3 H C3 H

C3 H

ACTIVITY,

4° 65 60 75

Number Revertants Of per Io 6 revertants survivors i~l Y . o ~ l

8. 7 I6.6 I4.t 16.3

2.66 4.66 3.53 3 .88

t26 I38 I32 136

300 Io3o 465 757

i3.8 42.9 2o.3 31.8

a n moles H C H O / m g protein/miu. All mice were males m a t c h e d for age and raised in the same e n v i r o n m e n t for at least two weeks prior to sacrifice, o.5 mg 3-nlethylcholanthrene in o.25 ml olive oil was injected i.p. 24 h prior to sacrifice; controls received o,25 ml olive oil i.p. Pools of 3 animals were used to determine each d a t a point. Microsomes were prepared according to the m e t h o d of KUPFER AND LEVI N{I; protein was measured by the Lowry techniqueL Identical 15-rain incubations were clone for formaldehyde production and bacterial mutagenesis except no bacteria were present in the demethylase assay. Incubation contained ioo m M DMN, 1.6 nlg microsomal protein per ml, r.6 mg T P N H per ml, p H 7-5 buffer, and either water or S. t y p h i m u r i u m in o.9°/.,,, saline.

i n c r e a s e d a c t i v a t i o n of D M N b y b o t h s t r a i n s , b u t t o d i f f e r e n t d e g r e e s . F o r m a l d e h y d e , a s i d e p r o d u c t of D M N a c t i v a t i o n , w a s a l s o m e a s u r e d 1°, a n d c o r r e l a t e d w e l l w i t h t h e i n d u c t i o n of h i s t i d i n e r e v e r s i o n s (Fig. I a n d T a b l e I X ) . T h e r e f o r e , t h e i n i t i a l d e m e t h y l a t i o n of D M N is p r o p o r t i o n a l t o i t s a c t i v a t i o n t o a m u t a g e n , a n d D M N d e m e t h y l a t i o n is p r o b a b l y t h e r a t e c o n t r o l l i n g s t e p i n t h e a c t i v a t i o n of D M N t o a m u t a g e n . However, alternative metabolic pathways proportional to demethylation have not been ruled out.

379

MICROSOMAL MUTAGENICITY ASSAY 5¢

pC57BL/6 / 3-MC . / induced

4£

II/C3H f 3-MC induced

.>>o_30 e~

% E o

3H

2C

~/C57BL/6

rr 10

I

r

I

J

50 100 150 200 n moles formaldehyde Fig. i. Relationship of DMN demethylase activity to DMN mutagenicity in two inbred mouse strains with and without 3-methylcholanthrene induction. Data are from Table VII. R E F E RENCES I AMES, B. 1~., et al., Carcinogens are mutagens. A simple test system combining liver homogenates for activation and bacteria for detection, Proc. Natl. Acad. Sci. USA, 7° (1973) 228i. 2 AMES, B. N., F. D. LEE AND W. E. DURSTON, An improved bacterial test system for the detection and classification of mutagens and carcinogens, Proc. Natl. Acad. Sci. USA, 7° (1973) 782-786. 3 BRUSICK,D., AND H. ANDREWS,A comparison of the genetic activity of chemical mutagens in Saccharomyces cerevisiae strains D3, D 4 and D 5 utilizing in vitro assays with and without hepatic enzyme activation. Environmental Mutagen Society, Washington, D.C. (1974). 4 DRUCKREY, H., et al., Chemische Konstitution und carcinogene Wirkung bei Nitrosaminen, Naturwiss., 48 (1961) I34. .5 FELLER, D. R., M. MORITAAND J. P. GILLETTE, Enzymatic reduction of niridazole by rat liver microsomes., Biochem. Pharmacol., 20 (1971) 2o 3. 6 KUPFER, D., AND E. LEVINE, Monoxygenase drug metabolizing activity in CaCl~-aggregated hepatic microsomes from rat liver, Biochem. Biophys. Res. Commun., 47 (1972) 61 i. 7 LowRY, O. H., et al., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265. 8 MALLING, H. V., Dimethylnitrosaimne: Formation of mutagenic compounds by interaction with mouse liver microsomes, Mutation Res., 13 (1971) 425 . 9 MALLING, H. V., Mutagenicity of two potent carcinogens, dimethylnitrosamine and diethylnitrosamine, in Neurospora crassa., Mutation Res., 3 (1966) 537. lO MALLING,H. V., AND C. N. FRANTZ. I n vitro versus in vivo metabolic activation of mutagens, Environmental Health Perspectives, 6 (1971) 71. II MCCALLA,D. R., AND D. VOtlTSlNOS, On the mutagenicity of nitrofurans, Mutation Res., 26 (1974) 3. I2 MCGREGOR, D. B., Enhancement by rat liver preparations of the gene conversion frequency induced by ethylmethanesulfonate in Saccharomyces cerevisiae, Mutation Res., 23 (1974) in press 13 MILLER, J. A., AND E. C. MILLER, Chemical carcinogenesis: mechanisms and approaches to its control, J. Natl. Cancer Inst., 47 (1971) v. 14 MOHN, G., AND J. ELLENBERGER, Mammalian blood-mediated mutagenicity tests using a multipurpose strain of Escherichia coli K-I2, Mutation Res., 19 (1973) 257-260. 15 NAKAJIMA,T., AND S. IWAHARA,Mutagenicityof dimethylnitrosamine in the metabolic process by rat liver microsomes, Mutation Res., 18 (1973) 121. 16 ONG, TONG-MAN, (NIEHS, Research Triangle Park, N.C.) Personal Communication. 17 POPPER, H., et al., Mutagenicity of primary and secondary carcinogens altered by normal and induced hepatic microsomes, Proc. Soc. Exp. Biol. Med., 142 (1973) 727 • 18 WOLPERT, M. K., J. R. ALTHAUSE AND D. G. JOHNS, l~itroreductase activity of mammalian liver aldehyde oxidase, J. Pharm. Exp. Ther., 185 (1973) 202.