Influence of Microsomal and Cytosolic Fractions from Rat ......absorbance of the product was determined in a Gilford 250 spectro-photometer (Gilford Instrument Laboratories, Inc.,

[CANCER RESEARCH 41, 4361-4367, November 1981]

Influence of Microsomal and Cytosolic Fractions from Rat, Mouse, and

Hamster Liver on the Mutagenicity of Dimethylnitrosamine in theSalmonella Plate Incorporation Assay1

Michael J. Privai2 and Valerie D. Mitchell

Genetic Toxicology Branch, Food ana Drug Administration, Department of Health and Human Services, Washington, D. C. 20204

ABSTRACT

Dimethylnitrosamine (DMN) was mutagenic in the Salmonellaplate incorporation assay (Ames test) at a level of 10 /Â¿mol/plate (3.7 HIM) in the presence of hamster liver S-9. Mutagenicity of DMN at this level was not observed when the S-9 was

derived from mouse or rat liver, although the mouse liver andhamster liver S-9 had similar DMN demethylase activities. Bothmouse and rat liver S-9 inhibited the mutagenicity of DMNmediated by hamster liver S-9; the inhibitory factor was contained in the microsomal fraction. Mouse or rat liver micro-somes did not inhibit the DMN demethylase activity of hamsterliver S-9. The microsomal inhibitor from rat or mouse liver wasstable at 60 but was inactivated at 70Â°.DMN demethylase fromboth rat and mouse liver was inactivated at 60Â°.Although the

DMN demethylase activity of hamster liver S-9 was contained

in the microsomal fraction, DMN mutagenesis under conditionsof the assay required the presence of both microsomal andcytosolic (S-105) fractions; the cytosols from hamsters, mice,

and rats were all effective. The cytosolic factor required forDMN mutagenesis was sensitive to trypsin and was not dialyz-

able.The presence of an inhibitor of DMN activation in rat and

mouse microsomes may account for, or contribute to, thefailure of liver S-9 preparations from these species to activate

DMN to a mutagen under standard conditions of the Ames test.The requirement for the cytosolic fraction may indicate thatDMN demethylase is not sufficient for the activation of DMN toa mutagen under the conditions used in these studies.

INTRODUCTION

The Salmonella plate incorporation mutagenicity assay(Ames test) is the most widely used method for screeningchemicals for potential carcinogenicity (31, 32, 38, 39, 41).The principal problem with this method (and other screeningtests) is the existence of "false negatives," that is, chemicals

that are carcinogenic when administered to animals but negative in the mutagenicity test. One of the most perplexing of theknown "false negative" chemicals has been DMN.3 This chem

ical is carcinogenic in a wide variety of animal species (22) butis negative in the standard Salmonella plate incorporationassay described by Ames ef al. (1 ) when rat liver is used as the

1A preliminary report of these results was presented at the 11 th Annual

Meeting of the Environmental Mutagen Society, March 17, 1980, Nashville,Tenn. (37).

2 To whom requests for reprints should be addressed.3 The abbreviations used are; DMN, dimethylnitrosamine; BSA, bovine serum

albumin; PBS, phosphate-buffered saline, [0.01 M sodium phosphate buffer (pH7.4X3.85% NaCI solution]; BAEE unit, AA253 of 0.001 /min with W-o-benzoyl-L-arginine ethyl ester as substrate at pH 7.6 at 25Â°.

Received February 23, 1981 ; accepted July 30, 1981.

source of S-9 for the metabolic activation system (7, 46). We

reported previously that DMN, at levels of 25 jumol/plate (9.3HIM) or higher, is mutagenic in this test if the S-9 is derivedfrom mouse or hamster liver (36). When hamster liver S-9 was

used, the mutagenicity of DMN could be observed at doses aslow as 1 /umol/plate (0.37 mM).

The current view of the pathway for the metabolic activationof DMN to the ultimate carcinogen or mutagen is shown inChart 1 (13, 34). This pathway involves only one enzymaticreaction, the hydroxylation of DMN by an NADPH-dependent,microsomal mixed-function oxidase. The assay for this enzymemeasures the formation of formaldehyde released as the hy-droxydimethylnitrosamine degrades spontaneously to mono-

methylnitrosamine. Thus, this enzymatic activity is generallyreferred to as DMN demethylase. Subsequent steps in theactivation of DMN are also viewed as occurring nonenzymati-

cally.The purpose of our studies was to determine if the varying

abilities of liver S-9 fractions from different species to activate

DMN to a mutagen in the Salmonella plate incorporation assay(Ames test) could be explained by differences in the DMNdemethylase activities of these S-9 preparations. Since both

mouse (3) and hamster (29) liver have been reported to havehigher DMN demethylase activities than does rat liver S-9, it

appeared that consideration of enzyme activities might besufficient to account for the inability of only rat liver S-9 toactivate DMN. However, the ability of hamster liver S-9 toactivate DMN at far lower DMN levels than the mouse liver S-9still required explanation.

MATERIALS AND METHODS

Chemicals and Media. "Gold Label" DMN (Aldrich Chemical Co.,

Milwaukee, Wis.) was used and was dissolved in sterile distilled water.PhÃ©nobarbital, BSA, acetyl acetone, trypsin (type XI from bovine pancreas), o-1-antitrypsin, and soybean trypsin inhibitor were obtainedfrom Sigma Chemical Co., St. Louis, Mo. Grade I yeast glucose-6-

phosphate dehydrogenase was obtained from Boehringer MannheimBiochemicals. Indianapolis, Ind. Nutrient broth from Baltimore Biological Laboratories, Cockeysville, Md., was used to prepare nutrient brothmedium as described by Ames ef al. (1 ). The PBS was 0.01 M sodiumphosphate buffer, pH 7.4, containing 0.85% NaCI solution, and thehomogenizing buffer was 0.1 M sodium phosphate buffer, pH 7.4,containing 0.25 M sucrose and 1 mM disodium EDTA. Both top agarand base agar were prepared as described by Ames ef al. (1) exceptthat the base agar contained 0.5% glucose rather than 2%. Aroclor1254, a mixture of chlorinated biphenyls, was a gift from the MonsantoCo., St. Louis, Mo.

Mutagenesis Assays. All mutagenesis assays were performed usingSalmonella typhimurium strain TA1530 (1 ); this mutant is derived fromS. typhimurium strain LT-2 which contains a base-pair substitution

mutation, hisG46, resulting in a requirement for histidine and a deletion

NOVEMBER 1981 4361

on July 4, 2021. © 1981 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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M. J. Privai and V. D. Mitchell

CH, CH CH CH,OH CH,

NIN

NAOPMOi N

NuO

CH X . N, CH,N,'OH

HCHO

Chart 1. Current view of the pathway of activation of DMN to a mutagen orcarcinogen.

through gal chi bio uvrB. The cells were grown overnight in nutrientbroth chilled on ice, washed once with PBS, and resuspended in PBSto a density of approximately 2 x 109 cells/ml. Mutagenesis assays

were performed by the plate incorporation method described by Amesef al. (1). The volumes of components in the top agar layer wereadjusted so that in all cases the final volume of the mixture poured onthe base agar was 2.7 ml. The cofactor mixture used to prepare S-9mix (or microsome mix) was as described by Ames ef al. (1) exceptthat the level of glucose-6-phosphate was increased from 2.5 to 10jumol/plate, and glucose-6-phosphate dehydrogenase was added at

1.4 units/plate. Colonies were counted with a Biotran II automaticcolony counter (New Brunswick Scientific Co., Edison, N. J.) unlessotherwise indicated.

Preparation of S-9 Fractions and Microsomes. Seven- to 8-week-old male Sprague-Dawley rats (ARS/Sprague Dawley, Madison, Wis.),

C57BL/6 x C3H F, mice (Charles River Breeding Laboratories, Inc.,Wilmington, Mass.), and Syrian golden hamsters (Charles River Breeding Laboratories) were used as the source of livers to prepare S-9fractions. Aroclor 1254 induction was performed as described by Amesef al. (1 ). To induce with phÃ©nobarbital, the drug was added to thedrinking water of the animals at 1 g/liter for 7 days before sacrifice.Food was removed from all animals on the day before sacrifice. S-9fractions were prepared as described by Ames ef al. (1) except thathomogenizing buffer was used rather than 0.15 M KCI. All S-9 preparations were stored at -80Â°.

For experiments involving uninduced, Aroclor-induced, and pheno-barbital-induced livers, 2 groups of animals (designated as "A" and"B" in Tables 1 to 3) for each species were used for preparing S-9.

Animals within each group were stratified by weight, and animals fromeach weight group were assigned to the uninduced, the phÃ©nobarbital,or the Aroclor group by using a table of random numbers.

The protein concentration of each batch of S-9 was determined bythe method of Lowry et al. (30) using BSA as the standard. Before eachexperiment, thawed S-9 was adjusted to a protein concentration of 30

mg/ml by dilution with homogenizing buffer.To prepare microsomes and cytosol, frozen S-9 was thawed and

centrifuged at 105,000 x g for 1 hr. The supernatant (cytosol) wascarefully removed. The pellet (microsomal fraction) was resuspended,centrifuged, and resuspended again in a volume of homogenizingbuffer equal to that of the original S-9.

Trypsin treatment of cytosol was performed by adding 4400 BAEEunits of trypsin to 0.9 ml of cytosol. After 12.5 hr of incubation, either72,000 BAEE units of a-1 -antitrypsin or 12,000 BAEE units of soybean

trypsin inhibitor were added.Enzyme Assays. DMN demethylase assays were carried out in 15-

ml conical centrifuge tubes. A cofactor mix was prepared so that theconcentrations of buffer and cofactors in the 2.7-ml enzyme assayreaction volumes were the same as those present in the 2.7-ml topagar mixtures used in the mutagenicity assays. The enzyme assayreaction mixture consisted of 0.3 ml of cofactor mix; 0.1 ml of DMNsolution; S-9, microsomal suspension, and/or S-105 as indicated for

the particular experiment; 0.2 ml of 0.1 M semicarbazide hydrochlorideneutralized with NaOH; and enough 0.5% NaCI solution to bring thefinal volume to 2.7 ml. All components except DMN were combined

and kept on ice. The reaction was initiated by adding the DMN andplacing the tubes in a New Brunswick rotary shaker bath at 37Â°shaking

at 200 rpm. After 40 min or less, the reaction was stopped by adding1.0 ml of 20% ZnSCX,, and 1.0 ml of saturated barium hydroxide wasthen added to precipitate the protein. The mixture was centrifuged, andthe formaldehyde produced by the DMN demethylase reaction wasassayed by the method of Nash (33) as follows.

Double-strength Nash Reagent B was prepared by adding 0.6 ml of

glacial acetic acid and 0.4 ml of acetyl acetone to 100 ml of 4 Mammonium acetate. To 0.8 ml of the double-strength Nash Reagent B,2.0 ml of the supernatant from the centrifuged enzyme reaction mixturewere added. The solution was incubated at 37Â° for 1 hr (10). The

absorbance of the product was determined in a Gilford 250 spectro-photometer (Gilford Instrument Laboratories, Inc., Oberlin, Ohio) at410 nm.

Under these assay conditions, DMN demethylase activity was linearfor at least 60 min and to at least 6 mg S-9 protein or its equivalentwhen microsomes were assayed. Added formaldehyde was found tobe stable under the enzyme reaction conditions, indicating that enoughsemicarbazide was present to efficiently trap and prevent degradationof enzyme-generated formaldehyde.

RESULTS

Comparison of Different Species and Inducers. Since it isgenerally believed that only a single enzymatic reaction isinvolved in the activation of DMN to a mutagen (Chart 1), weperformed experiments to determine whether differences in themutagenicity of DMN in the presence of S-9 from rats, mice,and hamsters could be explained by differences in their DMNdemethylase activities. Uninduced, phenobarbital-induced, andAroclor-induced liver S-9 fractions were prepared from 2

groups of each species of animal, designated A and B. Eachmutagenesis and enzyme assay was performed with 150 /il ofS-9 containing 4.5 mg of protein. The ability of each S-9 to

activate DMN to a mutagen and its DMN demethylase activitywere determined on the same day.

DMN demethylase from freshly prepared rat and mouse liverS-9 or microsomes has at least 2 forms with different apparent

Km values (3, 27). DMN demethylase I has an apparent Km inthe range of 0.2 to 1 mw (3, 21, 25, 40, 43). The apparent Kmof DMN Demethylase II has been reported to be in the range of35 to 130 mw (3, 11, 12, 25, 40). We found that our frozen S-9 preparations from rat, mouse, and hamster liver also have atleast 2 apparent Km values for DMN demethylase.4 In thepresent experiments, each S-9 was tested at 4 different dosesof DMN (3, 10, 100, and 540 /imol/tube) corresponding toconcentrations of 1.1, 3.7, 37, and 200 mM, respectively.Thus, at the 2 lowest DMN doses, the low Km form of DMNdemethylase should be primarily responsible for the metabolism of DMN; at the highest dose, the high Kmform, if present,should be active; and at 37 HIMDMN, both forms of the enzymecould contribute significantly to the metabolism of the substrate.

DMN Demethylase II was induced by both phÃ©nobarbital andAroclor, as shown by the increase in enzyme activity of inducedS-9 compared with uninduced S-9 at 200 mM DMN (Tables 1to 3). Comparison of the activities of S-9 from treated anduntreated rats and mice when 1.1 or 3.7 mM DMN was usedshows a repression of the activity of DMN Demethylase I byAroclor (Tables 1 and 2). Aroclor and phÃ©nobarbital treatments

' M. J. Privai and V. D. Mitchell, unpublished data.

4362CANCER RESEARCH VOL. 41



DMN Activation to a Mutagen by Liver Fractions

Table 1Mutagenicity of DMN activated by rat liver S-9 and DMN demethylase activities

of the S-9S-9 fractions were prepared from livers of 2 groups of rats (A and B) for each

type of Â¡nducertreatment. Plate incorporation assays for mutagenicity on S.typhimunum strain TA1530 were performed, and DMN demethylase activitieswere determined using 150 jil of each S-9 preparation containing 4.5 mg ofprotein.

Revertants/plate"DMN

(niM)1.11.11.13.73.73.7373737200200200InducerPBARPBARPBARPBARS-9.ANSl"NSINSINSINSINSINSI75NSINSI609805S-9.BNSINSINSINSINSINSINSI120NSINSI7061020DMN

demethylaseactivity(nmol/hr)S-9.

A103893713712755145315192259899609S-9.B91973212115237175269132215662361

'' Spontaneous revertan! counts (10 to 19) subtracted.

NSI, no significant increase (less than 10) over spontaneous revenant count:PB, phÃ©nobarbital;AR, Aroclor 1254.

Table 2Mutagenicity of DMN activated by mouse liver S-9 and DMN demethylase

activities of the S-9

S-9 fractions were prepared from livers of 2 groups of mice (A and B) for eachtype of inducer treatment. Plate incorporation assays for mutagenicity on S.typhimurium strain TA1530 were performed, and DMN demethylase activitieswere determined using 150 u\ of each S-9 preparation containing 4.5 mg ofprotein.

Revertants/plate8DMN

(mM)1.11.11.13.73.73.7373737200200200InducerPBARPBARPBARPBARS-9,ANSI0NSINSINSINSINSINSI36191NSI2262000S-9,BNSINSINSINSINSINSINSINSI96NSI831969DMN

demethylaseactivity(nmol/hr)S-9,

A1972367127130620029659895945413701860S-9,B2762386425730088294434437321609719

a Spontaneous revertan! counts (21 to 46) subiracted.6 NSI, no significant increase (less than 20) over spontaneous revertan! count;

PB. phenobarbÃ¼al;AR, Aroclor 1254.

appeared to have little or no effect on the activity of DMNDemethylase I in hamsters (Table 3).

The data in Tables 1 to 3 indicate that the ability of an S-9 to

mediate the mutagenicity of DMN was not simply a function ofits DMN demethylase activity. Uninduced or phenobarbital-induced mouse liver S-9 (Table 2) at the 1.1 and 3.7 mw DMNconcentrations had approximately as much DMN demethylaseactivity as did uninduced hamster liver S-9 (Table 3), but DMN

at these concentrations was mutagenic only in the presence ofhamster S-9. The failure of rat liver S-9 fractions to activate

DMN to a mutagen might be explained by their low DMNdemethylase activities (Table 1). However, the differences inthe ability of mouse and hamster liver S-9 preparations to

mediate the mutagenicity of DMN required some explanationother than DMN demethylase activity.

Mixing of Liver Fractions from Different Species. Since

uninduced mouse liver S-9 had approximately as much DMNdemethylase activity as did uninduced hamster liver S-9, and

since the product of DMN demethylase is thought to breakdown spontaneously in several steps to the ultimate mutagen,one possible explanation for our results might be that somecomponent of the mouse S-9 is capable of trapping one or

more of the intermediates in the pathway leading to the ultimatemutagen. If this were the case, then mouse S-9 might be

capable of inhibiting the mutagenicity of DMN mediated byhamster S-9.

When mutagenicity tests were performed with 3.7 mw DMNin the presence of 150 /il of hamster S-9 (4.5 mg of protein)and 150 /Â¿Iof mouse S-9, the mutagenicity of DMN was, in fact,inhibited (Table 4). Results obtained when mouse S-9 was

replaced with 150 pi of mouse cytosol or mouse microsomesindicated that the inhibitory factor was in the microsomal frac-

Table 3Mutagenicity of DMN activated by hamster liver S-9 and DMN demethylase

activities of the S-9

S-9 fractions were prepared from livers of 2 groups of hamsters (A and B) foreach type of inducer treatment. Plate incorporation assays for mutagenicity onS. typhimunum strain TA1530 were performed, and DMN demethylase activitieswere determined using 150 ,ulof each S-9 preparation containing 4.5 mg ofprotein.

Revertants/plate8DMN

(PIM)1.11.11.13.73.73.7373737200200200InducerPB6ARPBARPBARPBARS-9.A1611>20001356>2000>20001924>2000>2000>2000>2000>2000>2000S-9.B1491754286>2000>2000>2000>2000>2000>2000>2000>2000>2000DMN

demethylaseactivity(nmol/hr)S-9.

A1822422412583964173671010140068421103050S-9.B202189183276308386421844126060816802980

' Spontaneous revertan! counts (10 to 41) subtracted.

PB. phÃ©nobarbital;AR, Aroclor 1254.

Table 4

Effect of mixing hamster and mouse liver fractions on the activation of DMN to amutagen and on DMN demethylase activity

All fractions were obtained from uninduced animals; 150 ,ulof each fraction,corresponding to 4.5 mg of S-9 protein, were added unless otherwise indicated.The DMN concentration was 3.7 mM in all assays.

Uver fraction(s)addedHamsterS-9MouseS-9Hamster

S-9 + mouseS-9HamsterS-9 (300fil)HamsterS-9 -4-mousecytosolHamsterS-9 + mousemicrosomesHamsterS-9 + mouse microsomes (1 00/il)HamsterS-9 + mouse microsomes (50nl)HamsterS-9 + mouse microsomes (25ul)HamsterS-9 + mouse microsomes (10fil)Mouse

microsomesMousemicrosomes(60Â°)cHamsler

S-9 + mouse microsomes(60Â°)HamslerS-9 + mouse microsomes (70Â°)Revenants

plates"214610162524189014155715301847NDNO172209DMN

demethylase

activity(nmol/hr)328335536593ND6576NONDNDND2412330ND

Eight spontaneous revertan! counts not subtracted.6 ND. not determined.c Microsomes heated to 60Â°for 15 min." Microsomes heated to 70Â°for 15 min.

NOVEMBER 1981 4363




tion. When the quantity of mouse microsomes was reducedfrom 150 to 25 /tl or less, mutagenicity was no longer significantly inhibited. Although heating the mouse microsomal fraction to 60Â°for 15 min did not alter the inhibition of mutagenicactivity, heating to 70Â° eliminated the inhibition. Thus, theinhibitory factory was stable at 60Â°but not at 70Â°.

The DMN demethylase data shown in Table 4 demonstratethat the mouse microsomal fraction did not inhibit hamsterDM N demethylase activity. The enzyme activities of hamster S-

9 and mouse microsomal fractions were approximately additive. Mouse DMN demethylase was not the inhibitor, sincemouse microsomes heated to 60Â° retained their inhibitory

ability but had lost virtually all of their DMN demethylaseactivity. Furthermore, the heated mouse microsomal fractionhad no effect on the enzyme activity of the hamster S-9, again

demonstrating that the inhibition of mutagenesis was not operating through inhibition of DMN demethylase. When 300 julof hamster liver S-9 were present, mutagenesis was not in

hibited, showing that the inhibition was species specific andnot simply a result of having an excess of S-9, microsomes, or

protein present in the reaction mixture.Although the failure of rat liver S-9 fractions to mediate the

mutagenicity of DMN could be explained satisfactorily by theirlow DMN demethylase activities, we conducted an experimentto determine whether or not rat S-9 contained an inhibitor ofDMN mutagenesis similar to that in mouse S-9. The experiment

was identical to that presented in Table 4, except that rat liverS-9, microsomes, and cytosol were used. Results with rat liver

(Table 5) were similar to those obtained with mouse liver; thus,rat liver microsomes also contained an inhibitor of DMN-in-duced mutagenesis. This inhibitor was stable at 60Â°but not at70Â° and had no effect on the DMN demethylase activity ofhamster liver S-9. The inhibitor was not rat DMN demethylase,since this enzyme was unstable at 60Â°.

Effect of Cytosol on Mutagenicity of DMN. Because ourresults suggested that the activity of DMN demethylase (amicrosomal enzyme) did not always correlate with the ability ofan S-9 to activate DMN to a mutagen, we investigated whether,

under our assay conditions, hamster liver microsomes weresufficient to activate DMN to a mutagen. The data in Table 6show that, although almost all the DMN demethylase activity ofS-9 was recovered in washed microsomes, these microsomesdid not activate DMN to a mutagen unless the cytosol (S-105)was present. Cytosol had little or no effect on the DMN demethylase activity of the microsomes. The cytosol derived fromrat or mouse liver S-9 was approximately equally as effective

as that derived from hamster liver in stimulating the mutagenicity of DMN in the presence of hamster liver microsomes(Chart 2).

Heating the cytosol to 60Â° caused a loss of most of theactivator activity; at 70Â°,it was completely destroyed. Heating

at these temperatures caused a considerable amount of proteinprecipitation, so in these experiments, colonies had to becounted by hand rather than on an automatic colony counter.When BSA was added instead of cytosol, no mutagenic activitywas observed, excluding the possibility that cytosol activationis a nonspecific effect of soluble protein. When the quantity ofhamster liver microsomes used was increased from 150 to 200or 250 /J in the absence of cytosol, mutagenic activity was stillnot observed, indicating that the supernatant activation was

Table 5

Effect of mixing hamster and rat liver fractions on the activation of DMN to amutagen and on DMN demethylase activity

All fractions were obtained from uninduced animals; 150 Â»Iof each fraction,corresponding to 4.5 mg of S-9 protein, were added unless otherwise indicated.The DMN concentration was 3.7 rriM in all assays.

Liver fractionsaddedHamster

S-9RatS-9Hamster

S-9 + ratS-9HamsterS-9 (300>il)HamsterS-9 + ratcytosolHamsterS-9 + ratmicrosomesHamsterS-9 + rat microsomes (100/il)HamsterS-9 + rat microsomes (50Â¿il)HamsterS-9 + rat microsomes (25p))HamsterS-9 + rat microsomes (1 0fil)Rat

microsomesRatmicrosomes(60Â°)cHamster

S-9 + rat microsomes(60Â°)HamsterS-9 + rat microsomes (70Â°)dRevertants/

plate"2598122424412417107599211126182524NDND1132589DMN

demethylase

activity(nmol/hr)30870327565NDÂ°381NONDNOND480321ND

a Seven spontaneous revenant counts not subtracted.

ND, not determined.0 Microsomes heated to 60Â°for 15 min.a Microsomes heated to 70Â°for 15 min.

Table 6Requirement for cytosol in the activation of DMN to a mutagen

All fractions were obtained from uninduced animals; 150 ,/


LU

<_JO.

OCtuCLW

OCLU

LUOC

2500

2000

1500

1000

500

10 50 150

pii CYTOSOL ADDEDChart 2. Effect of different amounts of cytosol on the mutagenicity of DMN in the presence of 150 Â¿ilof hamster liver microsomes. Cytosols were from rat (A),

mouse (â€¢).and hamster (â€¢)livers. All S-9 fractions were adjusted to 30 mg protein per ml before preparation of cytosols and microsomes. Microsomes wereresuspended in the original S-9 volume. DMN concentration was 3.7 mM (10 fimol/plate).

Table 7

Effect of trypsin treatment on activity of the activator in hamster liver cytosolCytosol was pretreated as indicated at 37Â°for 12.5 hr. All mutagenesis assays

were performed on S. typhimurium strain TA1530 using 3.7 mw DMN, 150 i


induce DMN demethylase also increase the ability to activateDMN to a mutagen. However, the hypothesis that DMN activation to a mutagen is solely a function of DMN demethylaseactivity would require a correlation across species lines. Wefound that such a correlation does not hold under our testconditions.

Hutton ef al. (20), using microsomes from induced anduninduced Syrian hamsters of 6 different strains, observed asignificant correlation between DMN Demethylase I activity andability to activate DMN in liquid suspension. However, eventhough the overall correlation was significant, there were manyinstances in which an S-9 with more enzyme activity than

another resulted in fewer mutants.In these studies, Hutton et al. (20) found that 1 HIM DMN in

the presence of hamster liver S-10 or microsomes resulted in

significant mutagenesis. They had reported previously thatDMN was not active at this concentration when mouse liver S-

10 was used (21). Their results, obtained by using liquidsuspension treatment of bacteria, confirmed our finding of theability of hamster liver S-9 to activate DMN at lower concentrations than mouse S-9 in the plate incorporation assay (36).

Microsomal Inhibitor of DMN Mutagenesis. The fact thathamster S-9, but not mouse S-9, can activate low concentra

tions of DMN to a mutagen, even though the hamster DMNDemethylase I is no more active than the mouse enzyme, couldhave 2 possible explanations. Enzyme activities other thanDMN demethylase may contribute to the activation of DMN toa mutagen, or mouse liver S-9 may contain a factor that

prevents the DMN from expressing its mutagenic activity.Our mixing experiments (Tables 4 and 5) show that both

mouse and rat liver microsomes inhibited the ability of hamsterS-9 to activate DMN to a mutagen but had no effect on theDMN demethylase activity of the hamster S-9. Lotlikar ef al.

(28) also reported that mixing rat and hamster liver microsomesdid not cause inhibition of hamster DMN demethylase by therat microsomes. If hamster liver DMN demethylase is the principal enzyme responsible for activating DMN to a mutagen inour assays (according to the scheme in Chart 1), then mouseand rat liver microsomes must be capable of trapping one ofthe intermediates in the pathway following the demethylationstep. Alternatively, if hamster S-9 activates DMN to a mutagen

by a totally different pathway, then the mouse or rat microsomes may be inhibiting an enzyme, trapping an intermediate,or interfering with a necessary enzymatic activity in this otherpathway.

Cytosolic Activator of DMN Mutagenesis. Our finding thatcytosol, in addition to microsomes, is required for activation(Table 6) indicates the possible involvement of proteins otherthan DMN demethylase in the activation of DMN to a mutagenunder our assay conditions. This requirement for cytosol maybe dependent upon the protocol used for mutagenesis assays.Others (12, 15) have found that DMN was mutagenic to S.typhimurium in the presence of washed mouse liver microsomes that were free of cytosolic fraction. In those studies, thebacteria, DMN, and microsomes were incubated together inliquid suspension before plating.

Lake ef al. (27) reported that addition of cytosol from rat liverto the washed microsomal fraction resulted in a 2- to 4-fold

increase in DMN demethylase activity. Such an increase didnot occur in our experiments with hamster liver microsomes(Table 6). Lake et al. (27) also found that the activity of this

soluble enzyme activator was destroyed by boiling and waslost upon dialysis for 5 days. We found that the soluble factorrequired for DMN activation (but not affecting DMN demethylase activity) was destroyed by heating to 70Â°but was not lost

upon dialysis for 24 hr.The effects of cytosol in increasing DMN binding to protein

(16) and DNA (24) and/or mutagenicity of DMN (Table 6) maybe due to the presence of activating enzymes other than DMNdemethylase in the soluble fraction. Alternatively, the increasesmay be due to some other effect of the soluble fraction, suchas stabilization of the microsomal DMN demethylase (27).

Hecker ef al. (18) found that adding cytosol to a liquidsuspension mutagenesis assay of W-nitrosopyrrolidine containing rat liver microsomes resulted in an 8- to 10-fold enhancement of mutagenic activity. They postulated (19) that the supernatant speeds activation of the nitroso compound by removing the product of the first step in the activation pathwayand accelerating its activation to a mutagen. Terriere and Chan(42) have reported that the supernatant contains a solublefactor that enhances the microsomally mediated A/-demethyla-

tion of ethylmorphine. Unlike the factor we were working with,however, this factor was stable to boiling.

Implications for Screening. Since the Salmonella plate incorporation assay is widely used to screen chemicals for potential carcinogenicity, the fact that some chemicals are mutagenic in this assay only in the presence of S-9 from certain

species raises an important practical question concerning thesuitability of screening all chemicals with only one type of S-9.We reported previously that hamster S-9 is more effective thanis mouse S-9 and that mouse S-9 is more effective than is ratS-9 for detecting the mutagenic activity of diethylnitrosamineand N-nitrosodi(n-butyl)amine (36). Bartsch ef al. (9) reportedthat bis(2-hydroxy-n-propyl)nitrosamine and methyl-n-propyl-nitrosamine are mutagenic in the presence of phenobarbital-induced hamster liver S-9 but not rat liver S-9. Hamster liver S-

9 was also found in a collaborative study to be capable ofactivating p-rosaniline, while rat liver S-9 was not (14). Although

there are not as yet enough data available to recommend theuse of hamster liver S-9 for general screening rather than thestandard Aroclor-induced rat liver S-9 recommended by Amesef al. (1 ), hamster liver S-9 should certainly be used to test anyW-nitroso compound that is negative in the presence of rat liverS-9.

Rat or mouse liver S-9 interferes with the mutagenicity ofDMN mediated by hamster liver S-9. We therefore concludethat it is not advisable to use mixed rat and hamster S-9preparations for screening chemicals as has been recommended by Weinstein ef al. (45).

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NOVEMBER 1981 4367



1981;41:4361-4367. Cancer Res Michael J. Prival and Valerie D. Mitchell Assay

Plate IncorporationSalmonellaDimethylnitrosamine in the Mouse, and Hamster Liver on the Mutagenicity of Influence of Microsomal and Cytosolic Fractions from Rat,

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