rat hepatocyte assay for DNA damage: improved criteria for assessment of cytotoxicity and genotoxicity and results for 81 compounds

Genetic Toxicology

ELSEVIER

Mutation Research 368 (1996) 59-101

Revalidation of the in vitro alkaline elution/rat hepatocyte assay for DNA damage: improved criteria for assessmentof cytotoxicity and genotoxicity and results for 81 compounds Richard D. Storer * , Troy W. McKelvey, Andrew R. Kraynak, Michael C. Elia ‘, John E. Barnum, Lori S. Harmon, Warren W. Nichols, John G. DeLuca of Genetic

Depurtment

cud

Cellultrr

Received

Tmicolo~~,

I February

Merck

1995; revised

Resrtrrc,h

Lahornrorie.s.

5 September

WP45-311,

1995: accepted

Wart Point,

5 September

PA 19486,

USA

I995

Abstract The in vitro alkaline elution/rat hepatocyte assay is a sensitive assay for genotoxicity, measured as DNA strand breaks induced in primary cultures of rat hepatocytes after 3-h treatments with test compounds. Since DNA degradation can be rapid and extensive in dead and/or dying cells, the original criteria for a positive result in the assay were that a compound induce a 3.0-fold or greater increase in the elution slope (for the terminal phase of alkaline elution from 3 to 9 h) in the absence of significant cytotoxicity (defined as relative cell viability of less than 70% by trypan blue dye exclusion; TBDE). Recently we have shown that false-positive results can still be obtained due to cytotoxicity when loss of membrane integrity is a late event in toxic cell death relative to the induction of endonucleolytic DNA degradation. To improve the ability of the assay to discriminate between genotoxic vs. cytotoxic effects of chemicals. we have evaluated additional assays of cytotoxicity including cell adenosine triphosphate (ATP) and potassium (Kf) content, tetrazolium dye reduction (MIT), TBDE after a further 3-h recovery incubation without test chemicals (delayed toxicity), cell blebbing and endonucleolytic DNA degradation (double-strand breaks: DSBs) assessed by pulsed-field gel electrophoresis (PFGE). We have also

Abbreviations:

AFB,.

aflatoxin

B,;

AAF

or 2-AAF,

2.acetylaminotluorene:

anisole; BLEO, bleomycin: BPA, bisphenol A: CA, chromosome aberrations: ovary:

CM,

chlorpheniramine

2,4-dinitrophenol;

maleate;

2.4.DCP.

2.4.dichlorophenol:

DMS,

ATP. adenosine triphosphate; BHA. butylated hydroxCAS. chemical abstracts service: CHO, Chinese hamster dimethyl sulfate: DMSO, dimethyl sulfoxide; 2,4-DNP,

DSB, double-strand breaks: EE, equivocal evidence: EMS, ethyl methanesulfonate; GA, geranyl acetate; GalN, Gy, gray: IA, iodoacetate: kb, kilobase( LIND, lindane: M, DNA molecular weight markers: ML, mouse lymphoma

Bgalactosamine; L5178Y mammalian cell mutagenicity assay: MTT. thiazolyl blue (3-(4,S-dimethylthizol-2-yl]2.5-diphenyl~e~razolium tive: NaF. sodium fluoride; ND, not done/no data: NE. no evidence: N-OH-AAF, ,v-hydroxy-2.acetylaminofluorene; Toxicology Program: PACE, programmed autonomously controlled electrode: PB. phenobarbital: PFGE, pulsed-field

bromide); N, negaNTP, US National gel electrophoresis;

PHEN. phenanthrene; ROTE, rotenone: SA, Salmonella microbial mutagenicity assay; SCE. sister chromatid exchange; SDS, sodium dodecyl sulfate; STZ. streptozotocin; TBDE. trypan blue dye exclusion viability synthesis; WY, Wyeth 14,643 I Corresponding author. Tel.: (215) 652.5872; Fax: (215) 652.7758. ’ Present address: Dr. Michael C. Elia. Astra-Merck, 725 Chesrerbrook Blvd.. 01651218/96/Sl5.00 SD1 0 165-12 18(95

0 1996 Elsevier )00070-5

Science

B.V.

All rights

reserved

assay

Wayne.

; TET.

tetracycline-HCI:

PA 19087.5677.

UDS,

unscheduled

DNA

evaluated 2 parameters derived from the elution data which can indicate extensive. cytotoxicity-induced DNA degradation: the fraction of the DNA recovered in the neutral lysix/rinse fraction and the l-intercept of the extrapolation of the 3-9-h segment of the elution curve. Twenty-eight rodent non-carcinogens that are negative (or inconclusive) in the Amesassay with no, or limited, other evidence of genotoxicity. and 33 genotoxins, most of which are also carcinogens. were evaluated. The results showed that DNA degradation as measured by a I-h PACE (ProgrammedAutonomously Controlled Electrodes)/PFGEassaywasa sensitiveindicatorof cytotoxicity whichcorrelated well with results of the other cytotoxicity indicators.The delayedTBDE (after a 3-h recovery), intracellular potassium and ATP assaysas well as the ?-intercept parameterwerealsoshownto be sensitiveand in some cases complementarymeasures of cytotoxicity. Usingnew criteria

basedon thesedataof an inducedslope(treatmentslope-negative controlslope)of 0.020for the 3- to 9-h elutionperiodand cytotoxicity limits of 70% relative viability for the delayedTBDE assayandSO7rfor intracellularATP content,the assay scoresthe genotoxicity of these61 referencecompoundswith an overall accuracyof 92%. Test results using these new criteriaare providedfor an additional20 compounds (5 non-genotoxiccarcinogens and I5 compounds whosegenotoxicand carcinogenicpotentialare unknownor equivocal). Kr~~rords: Alkaline elunon: Genotoxicity: Cytotoxicity: Rebalidation

1. introduction Short-term in vitro tests for genotoxicity play an important role in the initial screening of new pharmaceutical candidates for their potential to induce mutagenic and/or carcinogenic effects. In our laboratory, an in vitro alkaline elution assay in primary cultures of rat hepatocytes is run routinely as part of the in vitro genotoxicity test battery. This assay sensitively detects DNA single- and double-strand breaks caused by physical, chemical and/or enzymatic attack. DNA strand breaks may be induced directly, by DNA excision repair enzymes, through alkali-labile lesions that result from chemical adduction of nucleotide bases or of the phosphodiester backbone by test chemicals. their metabolites or oxygen radicals, or through inhibition of enzymes such as topoisomerases.Since cytotoxicity can also result in the production of DNA strand breaks due to activation of degradative nucleasesin dead and/or dying cells (Bradley et al., 1987; Kohn. 1991; Elia et al., 1991, 1993, 19941,a careful assessmentof cytotoxicity and utilization of well-validated cytotoxicity limits are important determinants of the assay’s specificity in discriminating between genotoxic and non-genotoxic (but cytotoxic) chemicals. Initial validation studies with the in vitro alkaline elution rat/hepatocyte assay showed that the assay was highly accurate as a predictor of the mutagenic and carcinogenic activity of a diverse set of 91 compounds(64 carcinogensand 27 non-carcinogens) from different chemical classes(Sina et al., 1983).

The criteria that were developed for scoring the assay basedon this data set were that a compound would be classified as genotoxic if it produced a 3.0-fold or greater increasein the elution slope (rate> at dose level(s) with greater than 70% cell viability relative to control as measuredby a trypan blue dye exclusion assay at the end of the 3-h treatment period (TBDE-0). The elution slope was defined as the elution rate for the terminal phase(from 3 to 9 h) of the alkaline elution step. Increasesin the initial rate of elution (from O-3 h) that are associatedwith rapid elution of degraded DNA from dead and/or dying cells are not therefore included in the elution slope value (Sina et al., 1983; Kohn. 1991). During the 12 years that this assay has been in routine use in our laboratory. two problems with these criteria have become apparent that led us to undertake a seriesof revalidation experiments. First. we have found, asrecently reported by Kohn (1991). that criteria based on a fold-increase in the elution slope are very sensitive to variations in the concurrent control slope, especially when the concurrent control slope is very low. Secondly, we have found. as recently reported by Elia et al. (1993, 1994), that in a few casesthe TBDE-0 assay is not an adequate measureof cytotoxicity. Significant endonucleolytic degradation of DNA in dead and/or dying cells leading to increasesin the elution slope can occur at doselevels of somecytotoxic compoundsthat do not affect trypan blue exclusion until sometimeafter it is usually measured(at the end of the 3-h treatment). Thus, cytotoxicity that is not adequately measuredby

the dye exclusion assay can confound interpretation of the data and lead to false-positive results. We have undertaken the revalidation study reported here with the objectives of (1) determining the appropriate means of assessing the cytotoxic effects of chemical treatments in primary rat hepatocyte cultures and (2) developing criteria which utilize an ‘induced’ slope parameter (treatment slope minus the concurrent negative control slope) instead of a fold-increase parameter. To accomplish these objectives, we chose an empirical approach of testing a large number of compounds which have been well characterized as to their genotoxic and/or carcinogenic potential. The test plan included running a conventional alkaline elution assay (with measurement of the fraction of DNA recovered from the neutral lysis/rinse steps) and performing the following assays for cytotoxicity on samples of the same treated cells as used for alkaline elution: the conventional trypan blue dye exclusion assay performed after the 3-h treatment (TBDE-0). trypan blue dye exclusion performed after replating treated cells in normal, compound-free medium and incubating them for an additional 3 h to allow for recovery from cytotoxic injury (TBDE-3), cellular adenosine triphosphate (ATP) levels and potassium (K+) content, tetrazolium dye (MTT) reduction, light microscopic evaluation of cell blebbing and DNA degradation (double-strand breaks) measured by pulsed-field gel electrophoresis (PFGE). The test plan also included evaluation of 2 parameters derived from the elution data which can indicate extensive, cytotoxicity-induced DNA degradation: the percent of the DNA recovered in the neutral lysis/rinse fraction and the y-intercept of the extrapolation of the 3-9-h segment of the elution curve. Data from these studies allowed us to establish new guidelines for cytotoxicity assessment in the assay and to improve the criteria for evaluating the genotoxic activity of test compounds in the assay.

2. Materials

and methods

2. I. Chemicals All test agents were obtained from Sigma-Aldrich Corporation (Sigma Chemical Co., St. Louis, MO

and Aldrich Chemical Co., Milwaukee, WI) except for bleomycin (BLEO; Fluka Chemika-Biochemika, N-hydroxy-2Buchs, Switzerland) and acetylaminofluorene (N-OH-AAF; Chemsyn Science Laboratories, Lenexa, KS). All test compounds and reagents were of the highest purity commercially available. 2.2. Isolation, culture and treutment of primary hepatocytes

rut

Primary rat hepatocytes from anesthetized (sodium pentobarbital, 1 ml/kg of a 1 grain/ml solution given i.p.) male Sprague-Dawley rats (150-300 g, Charles River Laboratories, Raleigh, NC) were prepared by a two-step EGTA/collagenase perfusion of the liver as previously described (Bradley et al., 1982; Bradley and Sina. 1984). The isolated cells were resuspended in 15-30 ml of Hank’s balanced salt solution buffered with 10 mM Hepes (pH 7.4). To remove dead cells, 5 ml of the cell suspension was gently layered onto 10 ml of cold 40% Percoll (Pharmacia. Piscataway, NJ) in Dulbecco’s phosphate-buffered saline with 10 mM Hepes (pH 7.4) in 30 ml polycarbonate tubes and centrifuged at 20000 X R for 10 min at 4°C. The bottom layers of cells from the Percoll gradients were removed, pooled, and resuspended in Leibovitz’s L- 15 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, 100 pg/ml streptomycin, 20 mM Hepes and 0.2% sodium bicarbonate. After a hemacytometer cell count and viability check by trypan blue dye exclusion, cells were diluted to 5.2 X IO’ cells/ml. The initial viability of the hepatocyte preparations used ranged from 8 l-97% (mean i S.D. = 88.7 + 4.6% for 29 assays) with the minimum initial viability taken as 80%. To initiate the exposure, 1 ml of cell suspension was added to each 100 mm microbiological dish containing 12 ml of culture medium after addition of test compounds for a final cell density of 4 X IO5 cells/ml. All exposures were for 3 h at 37°C under 5% CO,. Test compounds were initially dissolved in dimethyl sulfoxide, water or acetone at 100 times (for compounds in DMSO and acetone) or 20-100 times (for compounds in water) the final concentration used in the culture medium. Cells harvested from untreated control plates at the end of the 3-h incubation and

irradiated on ice with 40 Gy of gamma radiation served as a positive control for double-strand breaks in the pulsed-field gel electrophoresis assays. Cells treated with 0.2 PM aflatoxin B, for 3 h or irradiated with 3 Gy of gamma radiation on ice served as positive controls for the alkaline elution assays. All chemical treatments were carried out in duplicate plates and solvent negative controls were run in quadruplicate plates. The highest concentration tested was 10 mM, as previously recommended for testing of soluble materials in mammalian assays (Kirkland. 1994). The solubility of test compounds in the culture medium was checked by dark-field microscopic examination of culture media treated with the highest concentrations of the test compounds at the end of a 3-h incubation. When compound precipitation is detected, alkaline elution results are not considered reliable since (1) precipitate can clog the pores of the elution filters creating problems at the stage of elution column loading, (2) phagocytosis of precipitate may lead to intracellular concentrations of drug which are exceedingly high ( > 10 mM) and unknown in magnitude, and (3) precipitate may induce cytotoxicity and/or DNA fragmentation as a consequence of the physical properties of the precipitate. Artifactual results might also be obtained if precipitate is trapped by and remains on the elution filter with the lysed DNA and DNA strand breaks are subsequently induced only during the 9-h elution step by DNA-reactive degradates of the test compound formed only at the very alkaline pH ( 12.1) of the eluting solution. 2.3. Alkaline elution assays Alkaline elution assays were performed as previously described (Elia et al., 1993) with several modifications. Briefly, 1.O X 10” cells on ice were loaded in chilled elution filter support columns containing 2.0 pm polycarbonate filters and lysed for 30 min with 2.0 ml of lysis solution (2% SDS, 50 mM Tris, 50 mM glycine, 25 mM Na,EDTA, pH 9.6 plus 0.5 mg/ml proteinase K). After 30 min, the lysis solution was allowed to drip out into a collection vial and 1.5 ml of a rinse solution (5 mM Tris, 5 mM glycine, 25 mM Na,EDTA, 50 mM NaCl, pH 9.6) was added, allowed to drip through and collected in the same vial. The pH 9.6 lysis and rinse solutions

from each elution column were analysed as the first fraction in order to measure the extent of DNA double-strand breaks associated with DNA degradation in dead and/or dying cells. Following addition of 30 ml of alkaline elution buffer (2% tetrapropyl ammonium hydroxide, 0.2% SDS. 20 mM H,-EDTA, pH 12.1) to the column, 3 fractions of 3 h each were collected at a flow rate of 0.035 ml/min. The fifth fraction consisted of the approx. 2.3 ml of solution remaining in the lines at the end of the 9 h elution period plus 4.0 ml of 0.4 N NAOH pumped through the column filter support and elution lines to remove any DNA remaining after removal of the elution tilter and reassembly of the columns. The sixth ‘fraction’ consisted of the DNA remaining on the elution filter at the end of the 9 h of alkaline elution. The DNA in fractions l-5 was then precipitated for 2-3 h at - 20°C after addition of 2 vols of 95% ethanol/O.25 M potassium acetate acidified with 6 ml/liter (for fractions l-4) or 14 ml/liter (fraction 5) of glacial acetic acid. The DNA remaining on the elution filter (‘fraction’ 6) and precipitated DNA from fractions 1-5 were quantitated fluorimetrically as previously described (Bradley and Sina, 1984) using the diaminobenzoic acid dihydrochloride (DABA) reaction (Kissane and Robbins, 1958). Fluorimetric readings were obtained using a Perkin Elmer Model 650-40 spectrofluorimeter at an excitation wavelength of 415 nm and an emission wavelength of 510 nm. Solutions of calf thymus DNA spotted and dried on prewashed (by trap filtering an ethanol-acetate/elution buffer solution) filters served as DNA standards for the fluorimetry. The elution slope values were calculated from semi-logarithmic plots of the fraction of the total DNA retained on the elution filter (log axis) versus elution time (linear axis) and are expressed as the absolute value of the average rate of elution per 3-h fraction for the terminal phase (3 to 9 h) of alkaline elution, as previously described (Sina et al., 1983). As suggested by Kohn ( 199 1). data are presented as ‘induced’ slope values, i.e., the arithmetic difference between the elution slope value for a treatment dose level (the mean slope value for duplicate plates) and the elution slope for the appropriate solvent control (the mean slope value for quadruplicate plates). The v-intercepts of the 3- to 9-h elution curves were determined by linear regression from the least-squares

line of best fit for the log of the fraction remaining after 3, 6 and 9 h of elution.

DNA

Irreversible cytotoxic effects resulting in either an immediate or delayed loss of membrane integrity were determined by trypan blue dye exclusion (Sina et al., 1983) on cells harvested at the end of the 3-h incubation of hepatocytes with tests chemicals (‘immediate’ toxicity; TBDE-0) or on aliquots (0.8 X 106) of the treated cells which were centrifuged and replated in fresh medium without test compounds in 60 mm microbiologic dishes for an additional 3-h recovery incubation (‘delayed’ toxicity; TBDE-3). Absolute cell counts were performed as part of the TBDE-3 assay and monitored for selected cytotoxic compounds to insure that the total number of live and dead cells per plate was not reduced due to disintegration and/or lysis of dead cells. Cell blebbing was assessed by qualitative observations under light microscopy during the TBDE-0 viability determination. A (+) in the tables indicates a marked increase in the frequency of trypan blue negative cells with membrane blebs. Cellular ATP content per culture, as a percent of control, was measured by bioluminescence using a luciferin/luciferase assay, as previously described (Armstrong et al.. 1992: Elia et al., 1993). Intracellular potassium (K ‘) was measured by flame photometry using a Bachrach model 5 1 flame photometer (Bachrach Instruments, Pittsburgh, PA). Samples (1 X lOh cells) were washed twice with 5 ml of cold. potassium-free. Dulbecco’s phosphate buffered saline (pH 7.4) and the dried cell pellet extracted with 0.5 ml of 107~ perchloric acid. Supematants were then introduced into the flame photometer which was calibrated using standard solutions of potassium chloride. Tetrazolium dye reduction (MTT assay) was measured in triplicate for each hepatocyte culture in a microtiter plate assay using I X IO’ cells/well incubated for an additional 2 h in 200 ~1 of medium at 37°C under 5% CO, in the presence of 500 pg/ml of MTT (thiazolyl blue: 3-[4,5-dimethylthizol-2-yl]-2,5-diphenyltetrazolium bromide). After spinning down cells, cell pellets were lysed in 200 ~1 of DMSO and the absorbance read at 570 nm in a model 7520 microplate reader (Cambridge Technology. Watertown. MA).

DNA double-strand breaks (DSB) were measured by I-h and 48-h programmed, autonomously-controlled electrode (PACE) pulsed-field gel electrophoresis (PFGE) assays. as previously described (Elia et al., 1993. 1994; Elia and Nichols, 1993). Scoring of the extent of DNA double-strand breaks produced was performed, as previously described (Elia et al.. 1994), by densitometric analysis and/or visual inspection of the amount of ethidium bromide-stained, high molecular weight (i.e., > 50 kbl DNA which accumulates in a narrow compression zone directly below the origin in the l-h PACE/PFGE assay. The 48-h PACE/PFGE assay was used primarily to assess the size distribution of the fragmented DNA. 2.6. Stmtistical u~u~!\..se~ The correlation calculations for the cytotoxicity parameters were performed using the correlation tool in Microsoft Excel Version 4.0 (Microsoft Corporation. Redmond, WA) and represent the covariance of the two data sets being compared divided by the product of their standard deviations.

3. Results and discussion 3. I. Compound selection and clu.s.s$kzTion In order to identify improved criteria for scoring the in vitro alkaline elution/rat hepatocyte assay, we sought to select for testing a large number of reference compounds for which there is sufficient data with which to classify the compounds into two groups: (I) rodent non-carcinogens which are negative in the Ames microbial mutagenicity assay with no or limited other evidence of genotoxic potential and (2) recognized genotoxins which. if tested for carcinogenicity, have also been found to be carcinogenic in rodents. Both of these groups of compounds include agents which also produce cytotoxicity after 3-h exposures in the dose range tested. The results from testing of compounds in these 2 categories were, therefore, judged to provide the most appropri-

64

I% DMSO 0.006

Geranyl acetate [ IOS-x7-31 (ML+, SCF.+W)

1% Water 0.010

glycol

[107-21-l]

Ethylene

0.20 0.25 0.30

0.05 0.10 0.15

7 I0

3

I .o 2.5 5.0

103 103 104 96 YX 96

0.000 0.000 -n.not - 0.002

97 02 99

100 91 95

I00

86

94 92

83 89 x2 86

92

II

100 66 17

104 96 X4

- 0.002 -0.001

- 0.002 ~ 0.003 - 0.007

-0.001 0.004 0.004(P)

- 0.003

0.5

I% DMSO 0.007

[536-33-41 (ML+)

~ 0.002

IO

Ethionamide

- 0.006 - 0.003

0.005 0.005 n.nos 0.0 IO

S.1 6.4 X.0 IO.0 3 7

I% Water 0.010

0.008

4. I

-0.OOI

Ephedrine sulfate [ 134-72-51 (SC%+ W)

(ML+)

sodium

I % DMSO 0.002

0. I73

0.8

Ditiocarh [20624-25-31

0.005 0.04 I 0.101

I

1% DMSO 0.01

2,4-Dichlorophenol 0.2 0.4 0.6

o.ow - 0.004

I 3 IO

I% DMSO 0.007

[ 120.83.21 (ML+, SCEf)

[94-20-21 (SCE + )

Chlorpropamide

I03 YS 9x 9s

100 100

99 Y4 101

98 101 95

YY

93 89

9.5

94 85 90 87 94

70

103 I03 XI

89

I OS 96

++ ++ ++ ++

-/(+) ++ ++

--/c+j + + +t ++

I05 107 102 IO1 loo YX

I20 Ill 122 II7 I20

I IX

122

YS

106 I08 104 100 10s

I08 I I6

63 7x 64

94

I05 II6

IO1

99 108 97 IO9 YY

72 3’)

95 49

108

94 I02

92 I03

IO1 IO4 78

IO1

100 86

III

104 100 IO2

9Y I0 I 76

YX

104 95

91

51

x4 102

85 81 78 70

31 32 I2

86

87

YY 97

97 Y4 86 77

84 72 42

95

91

++

++ (Yt ) ++

Yl x.3

+/c-j

I I7 III 126 124 I21

I I3

Y3

95 92

I ox I IS 96

IO5

I IO 91

I05 106 II5 II0 II3

Il.3

96 96 Y3

Y3 X6 35

Y4

84 79

88

35 24

66 65 I IO

53 45 41

22 I4 14

61

x3

97 04

95 91 83

3 3 2

52

56

95 x0

~ ~ +

-

~ -

~ -

~

-

-

+ +

+ + +

~ ~

-

-

~ ~

5% Water 0.004

Phenformin-HCI

[834-28-61 (ML?) 10

s 7

I 3

3.0

0.3 0.7 1.5

I% DMSO 0.012

p-Nitrophenol [ I Oo-02-71 (Cabs + )

0.07 0.10 0.15 0.21 0.30

I c/r DMSO 0.002

Methoxychlor [72-43-S]

0.3 0.7 I .o I .3

0. I

0.075 0.1 IO 0.145 0. I x0

0.040

0. IS

0.05 0.10

Dose(mM)

(ML+)

I % DMSO 0.002

I% DMSO

I% DMSO 0.004

Vehicle/ Elution slope h

Menthol [ 15.756.70.4]

.’

0.012

No.] results)

[434- 13.91 (MI.-+ +)

Lithocholic

(ML‘?)

I.indane [58-89-91

acid

I (continued)

Compound[CAS (NTP (ienetox.

Table

0.019 0.056 0.098 0.157

-0.001

68

89 71

97 96

78

96 96 Y8

97 107

9.5 97 04 91 93 84

101

-0.008 0.008 0.006 0.022

65 60

61 54

0.001(P) 0.004(P)

98 92 x2

88 58

YX 94 90

101 I03 91

97 92

84

94 95

% of control

Fraction DNA remaining after lysih rind rinse ’

98 91 81

67 40

0.008 0.01 I

”

0.00 I 0.00 I O.ooI(P)

96 89 87

0.005 0.005 0.008

0.024

104 102 62

~ 0.007 0.000

73 I05 97

(P)

93 93

~-intercept c/r of control

-O.OIO -O.OlO

0.009

0.002 0.003

Induced clution slope ’

douhlebreaks

(+) (+I (+) (+) (+ +)

(-) (+) (+I (+)

+ ++ ++

ND

+/c-j ++ ++ ++

(G) (+) (+) (+) (+)

i

DNA wand t

107 99 96 57 I3

94 85

81 64 31

98

67

85 86 60 60

61 30

99 95 73

63 43

IO1 94 x9

55

100 80

53 53

88 69 68

53 53 24

71

82 93

I I6 I IO 96

76 63

98 84 73

57 44 51

98 93

71 89 55

MTT

(% of control) TBDE-3

YX 99 87

38

77 68

91

90 78

107 98 10s

79 42

91 93 80

94 90 92 62

107

x.5

91 93

TBDE-0

Cytotoxicity

.L’

+ + +

94 Y3 84

3x 30

4 3

84 78 86 89 90

16 7 6 5

+

+ +

~ -

+ ~

+

+

94

80

~

+ + + 22

61 36

77

3 2 g 3

2 z

9 ST kc 5 2 : s

+

47

00 79 65

+ + +

96 83 66

2 z

-

101

$ 3 2 ;: r: 7

88 ~

-+

C’el blebbing

100 85 51

K+

26 4

54

98 88 90 71

35

104 104 105 77

26 13

IO.5 74 42

97 68

YY

ATP

SCE+)

Sulfisoxarole [ 127-69-S]

[10X-30-S]

Sucanic

Sodium [ISI-21-31

(ML+)

anhydride

dodecyl

sulfate

I% DMSO 0.007

I% DMSO 0.012

I% Water 0.00s

I7r DMSO 0.002

[88-96-O]

anhydridr

+ )

Phthalic [X5-44-91

+ SCC

- 0.00 I 0.000 - 0.002 - 0.002

I0

0.000 0.000 0.003

US

99 97 8.5

Yl Y2

97

4Y Y

0.0 I x 0.216

I0 I 103 92

YY Y7

YY 100

00 87 77

I

Yl Y.-i 91

90 90

84

86 X6

0.003 0.000 0.0 IO

0.005 0.002 -0.001

0.000 - 0.002 0,000(P)

- 0.00

0.00 I 0.003 0.003

-0.001 0.000

~ 0.004 0.004

~ 0.007

I 3 I0

3 7 IO

0.347

0. I39 0.191 0.233 0.2YS

I 3 I0

23 5.0

0.5 I .o

5.1 6.4 8.0 IO.0

I’% DMSO 0.007

Cabs

4. I

1% DMSO

IO

3 7

0.002

I% Water 0.0 IO

Phthalamide

(ML+,

Phenylthiocarhamidc [103-x5-51

(ML+,

Phenylephrine-HCI [6 I-76-71

++

no 60 64

X6

97 96 86

90 Y4

06

+/c-J

++

( -) (-) (4 (-)

++ ++

Y2 YO

101 103 93

YY 97

98 99

94 96 97

91 02

87 85

x9

YS 91

X5 Y3

106

92 I 09 106

98 06

91

I

88

96 94 88

I07 Y7

100

0

Yh 83 6X 36

97

Yl

Y4 81 77 44

96 YI

IO0 102

87 74 76

86 00

Y7 YI YO

94 04

102 IO2

100 100 x7

90 91

106

100 I 00

IO6 I02 98 99 89 77 77

I21 I32 10s 91 II3 106 I06

2

x4 77 20 7

XY 61 77 39

89 84

100

85

10s 101

100

95 92 80 X6

79 71

I OS 93 66 106 87 I IO

82 x4 xx

I I4 I I4 II3

99 I20 III

12’) 144 I44

100 IO0

96 102

I06

88 84

~ ~

-

~

~ ~

-

-

II2 10s

~ ~

+ + +

~ +

-

-

98 88 YX

74 73 69

89 x4

87

YO x5

\

‘, s ?

‘k oc

5 z f

2

B

I (continued)

in vitro

genetic

toxicology

wth

positive

exchange. its final concentration (vehicle) control

tests

I 2 4

0.2.50 0.313 0.39 I

0.200

Dose(mM) L

(P)

slope.

weak

positive

100 I 00 YS

92 88

9s Y4

(+W).

~~intcrcept ” % of control

in the media, and the elution (P). precipitate/insoluble

(+),

0.003 -0.001 ~ 0,00?(P)

0.002 0.000 0.001

0.002

\1ope

Induced clution

DNA after

inconclu~ivc

’

(‘!)

densitometrlc g Cytotoxicity (TBDE-3):

hrcaks (DSBs) : l-h PACE (Programmed produced by 40 Gy of gamma radiation. or (for scores in parenthe\e~) by visual

scores are listed for a particular dose Icvcl. assays: TBDE, trypan blue dye exclusion measured MTT. thiaroyl hluc dye reduction: ATP. intracellular

’ DNA double-strand DSBs lew than that dcnsitometric analysis immediately adenosine

Autonomously + +. DNA examination

’

Salmonella:

Y8 91 90

XI 54

95 102

TBDE-0

Cytotoxicity

vc. time)

Control

linear

ML,

89 85 85

X0 so

97 91

I06 96 YX

127 II0 120

values

ranged

or after a 3-h recovery potassium content.

incubation

to 9-h

to O.c)IS.

elution

in medium

without

compound

fragmentation. +. DNA Scores determined hy where both visual and

3

-

~ -

-

-

h’chhing

Ccl I

chromosome

0.735

95

96 Y3

85 62

Y9 99

K+

Cabs,

from

of the

lymphoma,

87 81 67

extrapolation

mouhr

x9

ATP

10s 94 78

g

IOX

MTT

(% of control) TBDE-9

Electrodes) pulsed-field gel electrophoresis assay for DNA to or greater then that produced by 40 Gy of gamma radiation. bromide stained gels. Data are considered marginal or equivocal

breaks).

of the

are listed.

SA,

(double-strand

filter

control

after the 3-h treatment (TBDE-0) triphosphate content: K +. intracellular

Controlled DSBs cqual of ethidium

doublebreakh

results:

-/(+)

-

+

+ + +

DNA strand

slope for the negativr dose level.

or

101 IOU 94

96 9s 92

97

lysis and rinse ‘/r of control

Fraction trcmaining

’ The y-intercept ia the intercept (on the v-axis of a wmi-log plot of fraction DNA remaining on the least-quare, line of best fit. Control values ranged from 0.702 to 0.905. ’ Fraction of total DNA remaining after the neutral lysia and rime steps, a measure of DNA degradation

aberrations; SCF.. \ister chromatld ’ The negative (vehicle) control, ’ Treatment slope minus negative

a NTP

If/r DMSO 0.007

Tolazamide [I 156.19-01 (SA’.‘)

h

0.006

slope

Vehicle/ Elution

[64-75-S] (ML+)

‘1

I% DMS’J

No.] rccults)

Tetracycline-HCI

Compound[CAS (NTP C;cnetox.

Table

$

?

2

k 53 2

$ z : a 2 2 z s

5 e.

Lr. 5 2

R.D. Storer et nl. /Mutatim

ate data set for an empirical selection of criteria for (I ) induced slope increases and (2) cytotoxicity limits for the assay which minimize false-positive and false-negative results and thereby maximize the accuracy of the assay in correctly identifying compounds with genotoxic potential.

Rrsrcrrch

69

368 (19961 59-101

3.2. Results,for non-curcinogenic compounds with no or limited ecidence qf genotoxicity The results for 28 compounds in the category of non-carcinogens with no or limited evidence of genotoxicity are presented in Table 1. All of these

B

Fig. I. Assay for DNA fragmentation (DNA DSB) by I h PACE/PFGE of several non-carcinogenic compounds with no or limited evidence of genotoxicity. Primary rat hepatocytea were treated with the indicated compounds for 3 h or with 40 Gy gamma irradiation on ice immediately prior to assay. Dose levels for each compound (see Table I) increase from left to right for (A) sodium dodecyl sulfate (SDS) and tetracycline-HCI (TET). (B) geranyl acetate (GA) and chlorpheniramine maleate (CM) and for (C) 2.4.dichlorophenol (2,4-DCP) and lindane (LIND). Dose levels for aflatoxin B, (AFB, ) and 2-acetylaminofluorene (AAF) controls were 0.2 PM and 5 PM. DNA molecular weight markers (MI are from 48.5 to 8.3 kb.

compounds were reported by the US National Toxicology program (NTP) as negative or, in several cases, inconclusive in the Salmonella microbial mutagenicity assay(s). With the exception of the detergent sodium dodecyl sulfate (SDS). they are also listed in the NTP data base as being negative (Nl for, or having no evidence (NE). of carcinogenicity in both sexes of rats and/or mice. We excluded from this group compounds which are reported as having equivocal evidence (EE) of carcinogenicity in either sex of rats or mice. For sodium dodecyl sulfate. results of the NTP genetic toxicology test battery were uniformly negative but the bioassay testing was discontinued. For approximately half of the 28 compounds in Table I, NTP results for one or more of the following in vitro genetic toxicology tests were reported as positive: the mouse lymphoma L5178Y mammalian cell mutagenicity assay (ML), sister chromatid exchange (SCE) in CHO cells and chromosome aberrations (CA) in CHO cells. Where this is the case. most commonly for the mouse lymphoma assay. the abbreviation for the positive in vitro test(s) is included in the Table I beneath the compound name and Chemical Abstracts Service (CAS) number. These data raise a question as to whether these compounds may properly be categorized as “nongenotoxic” compounds for the purpose of these validation studies. However. in our view, the absence of any carcinogenic effects of the compounds and the negative (or inconclusive) Salmonella microbial mutagenicity data suggest that the positive in vitro data for some, if not most, of these compounds may reflect indirect, cytotoxicity-mediated mechanisms of effects on genomic integrity rather than the direct and specific interaction of the compound or a metabolite with DNA or a DNA strand-breaking protein (e.g., topoisomerases). The data in Table 1 show that 7 of the 28 compounds (butadiene sulfone, butyl chloride, caprolactam, ethylene glycol, phthalamide, succinic anhydride and tolazamide) were essentially non-toxic at all concentrations tested up to IO mM or the limit of solubility. An additional 4 compounds (geranyl acetate. phenylephrine-HCl, phthalic anhydride and sulfisoxazole) produced only very weak evidence of dose-related cytotoxicity as judged by the more sensitive parameters of toxicity such as cell blebbing or

an increase in DNA degradation (double-strand breaks). None of these I I non-toxic or marginally cytotoxic compounds produced any consistent, dose-related increases in the induced elution slope. For the remaining 17 compounds which did show clear evidence of a dose-related cytotoxic effect by one or more of the parameters measured, 9 of the compounds also produced evidence of dose-related increases in the induced elution slope (chlorpheniramine maleate. 2.4-dichlorophenol, ditiocarb sodium, lindane, lithocholic acid. menthol. phenformin-HCll sodium dodecyl sulfate and pnitrophenol). While the effects on the induced elution slope were weak or marginal for ditiocarb sodium. D-menthol, and lindane, for the remaining 6 compounds there were moderate to large, dose-related increases in the induced elution slope (0.0220.216) which paralleled the dose-related increases in cytotoxicity and DNA degradation (double-strand breaks) seen by the I-h PACE/PFGE and/or by decreases in the fraction DNA remaining on the elution filter after the neutral (pH 9.6) lysis/rinse step. Representative examples of the l-h PACE/PFGE gels for several of these compounds are presented in Fig. I. Thus. the DNA strand breaks detected in hepatocytes treated with these compounds at cytotoxic dose levels include double-strand breaks and indicate extensive DNA fragmentation due to the activation of degradative endonucleases in dead and/or dying cells. For 4 of 6 compounds in Table I which produced moderate to large increases in the induced elution slope at cytotoxic dose levels (2.4-dichlorophenol. lithocholic acid. phenformin-HCI and I?-nitrophenoll. the sharp increases in the induced elution slope above background coincided with precipitous drops in cellular ATP levels and. with the exception of p-nitrophenol. with significant delayed toxicity as measured by the TBDE-3 recovery assay. The data for these compounds confirm our previously reported finding (Elia et al.. 1993) that cytotoxicity that is not adequately measured by the trypan blue dye exclusion assay performed immediately after harvest (TBDE-0) can be a significant confounding variable in the assay leading to false-positive results. For 2.4-dichlorophenol and phenformin-HCl for example. the increases in the elution slope are of sufficient magnitude that these compounds would have been

elution plot, but with minimal effects on the induced elution slope from 3 to 9 h. This phenomenon has previously been referred to by Kohn ( 199 1) as an ‘initial dip’ in the elution curve caused by rapid elution of degraded DNA from damaged cells or cells undergoing autolysis. The v-intercept parameter, however, also accounts for DNA eluted under ‘neutral’ (non-denaturing; pH 9.6) conditions from dead and/or damaged cells that is recovered in the lysis/rinse fraction prior to the alkaline elution step. Recovery of DNA in this lysis/rinse fraction may indicate near complete degradation to oligonucleoso-

considered positive in the assay by the original criteria (Sina et al., 1983) which used a 3.0-fold elution slope increase and 70% or greater relative cell viability by the TBDE-0 assay. Another potentially useful indicator of cytotoxicity is the v-intercept of the 3 to 9 h elution plots (illustrated in Fig. 2a). As shown in Fig. 2 for methoxychlor, D-menthol and chlorpheniramine maleate, most non-genotoxic compounds that are cytotoxic produce a change in the elution profile, a ‘parallel drop’, that is characterized by dose-dependent decreases in the v-intercept of the 3- to 9-h

Methoxychlor

Menthol

1.0

‘.OY

5 ir s P .cj 5 a

2 6 .-s 5 F L 01

0.1

3

0

6

9

3

0

Phenformin-HCI

6

Chlorpheniramine

9

Maleate n

P

0 0 A n

D

DMSO 1% 137~cs 4.4 G) 0.10 mM

0.25 mM 0 0.50 mM V 0.75 mM

0

3

Elution Fig.

2. Alkaline

evidence extrapolation

elution/rat

hepatocyte

assay

6

Time

for DNA

9

3

0

(Hours) strand

Elution break

induction

of genotoxicity (see Table I for elution slope and cytotoxicity of the 3 to 9 h elution plot back to the y axis) is illustrated

by several

data). in (A).

The

L.-I D

A l.OOmM

Time

6 (Hours)

non-carcinogenic derivation

of the

9

compounds x-intercept

with parameter

no or limited (a linear

72

R.D. Storer

et al. /Mutation

ma1 size fragments, as occurs in certain cell types undergoing apoptosis (Zwelling et al., 1991) but also reflects rapid elution from the filter of higher molecular weight (100 kb to 1 megabase), double-stranded DNA fragments produced by endonucleolytic DNA degradation in necrotic cells (Elia et al., 1993, 1994). Interestingly, for some compounds such as phenformin-HCl which is a mitochondrial toxin (Byczkowski et al., 1982), a parallel drop is not a characteristic feature of the cytotoxic response (See Fig. 2c and Table 1). We have seen similar results for potassium cyanide (see Table 9 and Fig. 10) which is cytotoxic due to its inhibitory effect on mitochondrial electron transport. For most of the compounds in Table 1 which produced some evidence of cytotoxicity, the viability measurements by trypan blue (TBDE-0 and TBDE-3) and the cytotoxicity assays (MTT dye reduction and cellular ATP and Kf content) were reasonably well correlated. For chlorpheniramine maleate, lindane, menthol, methoxychlor, phenylthiocarbamide, sodium dodecyl sulfate and tetracycline, for example, the viability measurements and results of the cytotoxicity assays showed very similar dose-related effects. For several compounds, most notably DLamphetamine sulfate, caprolactam and ethionamide, the MTT assay appeared to be a more sensitive indicator or the only indicator (for caprolactam) of dose-relatedcytotoxic effects but in no casewas this cytotoxicity accompanied by any significant increasesin the induced elution slope. In the case of ditiocarb sodium, methoxyclor and phenylthiocarbamide, however, the MTT assay was notably less sensitive than the other assays as a measure of cytotoxicity. Finally, it appears,as discussedabove, that for several of the compoundsin Table 1 which have the potential to produce increases in the induced elution slope (and false-positive results), cytotoxicity limits basedon the delayed toxicity (TBDE3) and ATP assaysare especially important. Observations of cell blebbing in trypan blue negative cells were also a sensitive indicator of cytotoxicity for somecompounds. The results of a pairwise correlation analysis of all of the data for the 28 compoundsin Table 1 for the 5 cytotoxicity assays, the y-intercept and lysis/drip fraction parametersfrom the alkaline elution data and DNA double-strandbreaks asmeasured

Resrurch

368 (19961 59-101

by the 1 h PACE/PFGE assayare shown in Table 2. The results show that the TPDE-3 assayhad the best overall correlation with the other 7 indicators of cytotoxicity followed closely by the TPDE-0 assay and the y-intercept parameter. The potassium, DNA DSB and ATP assaysalso showed a high degree of correlation with the other parameters. The MTT assayand lysis/drip fraction parameterhad the lowest degrees of correlation overall with the other measuresof cytotoxicity. Interestingly, the highest correlation (0.833) between any two assays/parameters was between the TBDE-3 and y-intercept values; the TBDE-0 and TBDE-3 assay values were also highly correlated (0.797). Thus, the y-intercept parameter, as suggested by Kohn (1991) for the initial ‘dip’ in filter elution curves, appearsto be a useful and reliable indicator of cytotoxicity which correlates well with cell viability assessed by trypan blue dye exclusion and measuresthe percentage of dead and/or dying cells in the treated population with extensively degraded DNA which elutes very rapidly either in the neutral lysis/rinse fraction or the 0- to 3-h fraction. 3.3. Resultsfbr genotoxins and genotoxic carcinogens The results for 33 compoundsin the secondcategory of genotoxins/genotoxic carcinogensare shown in Table 3. These include a diverse group of recognized genotoxic carcinogens and procarcinogens as well as compounds which specifically induce DNA strand breaks by mechanismsthat do not involve covalent binding of the test compound itself to DNA, such as inhibition of DNA topoisomeraseI or II, and which should therefore be detected as positive in the assay. Dose levels were chosen basedon published results or in a few cases, based on preliminary solubility and range-finding assays.In the latter case, the top dose level was chosen as a doseexpected to produce significant cytotoxicity or, in the absenceof any cytotoxicity, as the limit of solubility of the compounds in the culture medium or 10 mM, a recommended upper limit for the top dose when testing soluble materials (Kirkland, 1994). Assays were repeated as necessary when the initial dose range selection was too low or two high, when technical problems occurred or to clarify equivocal results at or near the limit of solubility.

0.61 I 0.724 0.772 0.833 0.605 0.707 5.049 0.72 I

4.910 0.701

0.791

TBDE-3

assays of 28 non-genotoxic

0.797 0.622 0.637 0.773 0.749 0.616 0.716

TBDE-0

for cytotoxicity

and/or

3.745 0.535

0.687 0.548 0.502 0.292 0.483

0.622 0.61 I -

MTT

4.241 0.606

0.637 0.724 0.686 0.68 I 0.596 0.299 0.618

ATP

non-carcinogenic

compounds

4.599 0.657

0.686 0.507 0.632

0.773 0.772 0.548 0.68 I

K+

4.869 0.696

0.749 0.833 0.502 0.596 0.686 0.8 I4 0.689

x-intercept

3.620 0.517

0.487

0.616 0.605 0.292 0.299 0.507 0.814

Lysis/Drip

” Correlation coefficients listed arc for the 24/28 compounds in Table I for which quantitative densitometric scores were available. ’ Sum is the sum of the correlation coefficienta for the cytotoxicity assay listed in the column heading with the other 7 assays or parameters. ’ Mean is the mean of the correlation coefficients for the cytotnxicity assay listed in the column heading with the other 7 assays or parameters.

Sum h Mean ’

”

coefficienta

TBDE-0 TBDE-3 MTT ATP K+ y-intercept I.ysis/Drip DNA DSB/PFGE

Table 2 Correlation

4.331 0.619

0.716 0.707 0.483 0.617 0.632 0.689 0.487

DNA

DSB/PFGE

results ”

Adriamycin-HCI [25316-40-91

Actinomycin [50-76-O]

D

2-Acetvlaminofluorene 53-96-31 :SA+,ML+,Cabs+,SCE+)

Compound [CAS No.] NTP genetox.

Table 3

0.008 0.018 0.083 0.288 0.573 0.003 0.001 0.000 0.00 I - 0.002 0.008 0.012 0.010 ND ND

1% Water 0.005

I % Water 0.005

I % Water 0.007

0.013 0.039(P)

1% DMSO 0.004

0.010 0.005 0.003 0.003

Induced elution slope ’

0.010 0.010 0.011 0.020

0.0010

Dose (mM)

I% DMSO 0.005

1% DMSO 0.00.5

Vehicle/ elution slope ‘I

87 93 96 ND ND

9.5 97 95 97 101

99 9s 109 91 96

102 99

102 IO5 95 93

98 96 94 96

?‘lnlcrccpt ” % of control

93 108 II5 ND ND

97 99 99 107 I I2

95 102 100 93 98

+ ++ ++ ++ ++ ++ ++ ++ +

99 I03 94 99 I06

-/(+)

96 102

96 94 I04 99

92 97 96 100

Cytotoxicity TBDE-0

IO0 100 103 95 95

-/(+I

99 97 IO1 93 98

-

100

-

C-1 C-J c--j (-)

DNA Double-strand breaks ’

103

96 96

102 103

98 97 9s 9.5

Fraction DNA remaining after lysis and rinse ’ % of control

95 101 IO1 100 99

96 92 89 95 100

loo 89 99 97 95

100 107

97 104 IO1 96

89 93 96 IO1

86 88 87 92 II2

III 91 94 III 98

83 101 98 82 95

99 73

86 95 86 82

I08 115 105 10s

99 98 87 8.5 91

92 IO6 93 99 91

100 85 I01 93 100

I28 120

94 140 99 95

IO7 118 105 I I2

(% of control) p TBDE-3 MTT .4TP

86 84 81 76 88

96 104 86 89 72

98 87 93 86 87

120 13.5

113 87 124 I16

II4 114 108 122

K ’

~ -

~

-

-

-

-

Cell blebbing

3 (continued)

Bleomycin sulfate [904 I-93-41

I % Water 0.009

0.0020 0.0066 0.0 I98 0.0463 0.066 I

0.0 I 0.03 0.07 0.10 0.15

I % Acetone 0.006

SCE+)

0.01 0.03 0.07 0.10 0.15

1% Acetone 0.009

Benzo[ alpyrene [50-32-g] (SA+, ML+.Cahs+.

I 3 6 IO

l%DMSO 0.003

0.17 0.2 I 0.26 0 32 0.40

O.OooO2 o.oooo5 o.ooo1o 0.00020

(mM)

Dose

Benzene [7 I-43-21 (SCE + )

l%DMSO 0.002

Azohenzene [103-33-31 @.A + , Cabs?/-SCE

+ /-)

1% DMSO 0.005

results ’

Vehicle/ elution slope h

Aflatoxin B I [I 162-65-81 (SA+)

Compound [CAS No.] NTP genetox.

Table

0.068 0.147 0.123 0.097 0.1 IS

0.001 0.010 0.017(P) 0.030(P) 0.0 I S(P)

0.001 0.006 0.014(P) 0.015(P) 0.026(P)

O.OOQ 0.000 0.001 0.002

0.009 0.013 0.020 0.149 ND(P)

0.004 0.019 0.034 0.096

Induced elution slope c

43 35 15 13 IO

100 99 100 102 98

101 107 IO1 99 98

102 101 99 98

58 49 49 10 ND

88 79 64 70

?‘lntcrccpt ’ % of control

78 78 67 62 53

100 99 99 100 98

102 107 IO1 99 97

101 IO1 99 98

76 70 81 66 ND

88 82 70 72

Fraction DNA remaining after lysis and rinse ’ % of control

+/c-j +/c-j ++ tt t+ ++ ++

t/C-)

ND ND + +

-

86 79 78 22 I7

+ +t ++ ++ ++

108 lo6 IO0 107 10s

101 99 98 102 IO1

102 I04 99 94 89

99 101 100 98

92 IO1 92 95

Cytotoxicity TBDE-0

(-) (-) (-) (6)

DNA Douhlc-strand breaks ’

I06 I06 loo 97 97

103 91 9.5 103 98

100 92 97 89 89

103 99 IO1 103

82 81 69 I6 3

93 I IO 86 I IO

I I3 88 87 75 84

93 I03 94 90 95

I I6 138 III I I8 124

96 86 I09 99

80 87 82 53 44

I08 93 IO1 I06

(% of control) E TBDE-3 MTT

88 III 87 87 89

96 96 74 100 80

98 II6 I IO 88 90

102 109 II3 I10

104 81 52 3 I

86 95 97 88

ATP

104 83 78 96 9s

I03 103 9s 102 I06

I04 II5 II9 96 90

98 88 89 97

85 85 69 I6 7

97 11s I08 102

K+

~

+

~ -

-

+ + +

-

Cell hlehhing

76

R.D. Storer

=0 8:

et al. /Mutution

Research

368 (19%)

59-101

F 5 I

I

I

I

I

I

I

I

I

I

I

++

I

++++

I

I

I

I

I

I

I

I

I

I

I

I

I

results ~’

SCE+)

Ethyl methanesulfonate [62-50-O] (SA+,Cabs+, SCE+)

Epichlorohydrin [ 106-89-81 (SA+,Cabs+. 1% DMSO 0.004

I% DMSO 0.005

I% DMSO 0.004

sulfate

Dimethyl [77-78-I]

0.30 1.00 3.00 10.00

0. IO 0.25 0.50 I .txl

0.03 0.10 0.30 1.00

0.01 0.03 0.07 0.10 0.15

0.03 0.10 0.30 1.00 3.00

1% Water 0.00.5

I% Acetone 0.006

)

0. I 0.3 1.0

0. I 0.3 0.7 I.0 3.0

1% Water 0.014

1% Water 0.003

0.03 0.10 0.30

Dose (mM)

1% Water 0.003

Vehicle/ elution slope h

7.12 - Dimethylbenz[a]anthracene [57 - 97-61 (SA+. ML+)

Dimethylnitrosamine [62-75-91 (SA + , CA?/-/ + , SCE+

Diethylnitrosamine [SS-18-S] (SA+)

~r-~

Comnound [CAS No.] NTP genetox.

Table 3 (continued)

0.015 0.065 0.475 0.433

0.00 I 0.015 0.234 0.324

0.01 I 0.101 0.663 0.173

O.COO 0.105 0.123 0.174 0.168(P)

0.04 I 0.06 I 0.100 0.129 0.173

0.048 0.047 0.08 1

0.06 I 0.088 0.133 0.155 0.328

0.018 0.019 0.03 I

induced elution slope c

98 109 250 41

96 93 109 II

95 100 155 7

97 103 77 84 75

I05 III I I6 123 120

I04 102 103

94 107 99 91 88

97 99 103

YIntercept “ % of control

-

98 99 96 93 93

-

97 97 99 99

(-) (-) (-) (+)

97 92 79 79

77

++

-

IO1 97

95 87 85

-

IO1 102 100

ND ND ND

-

98 102 96 96 101 95 96 93

ND ND ND

DNA Double-strand breaks ’

97 98 100

Fraction DNA remaining after lysis and rinse’ % of control

95 102 102 IO1

90 103 97 96

98 97 92 90

94 98 96 98 101

97 I01 I06 IO1 IOil

96 101 99

100 IO0 96 94 98

97 92 98

Cytotoxicity TBDE-0

100 96 I03 96

97 93 95 81

IO1 94 96 7s

9s 98 91 91 92

101 I04 100 IO1 IO1

97 93 I05

97 94 84 80 85

97 IO.7 89

70 78 83 68

91 98 98 82

82 91 95 61

77 I08 103 111 120

133 140 I08 I09 81

97 88 97

88 92 97 95 87

93 97 94

(8 of control) ?’ TBDE-3 MTT

97 77 108 II4

I is I06 99 96

103 I05 107 83

I IO 89 95 84 77

102 I06 96 IO1 107

10s loo I IO

83 97 108 I04 107

96 103 I09

ATP

78 79 84 84

107 II2 109 II0

92 85 76 5.5

127 103 10s 103 103

100 96 100 95 103

I06 109 102

IO1 II0 99 95 107

90 93 89

K’

-

~ -

~ ~

+ + +

~ -

-

-

~ -

Cell blebbing

78

R.D.

I 7 +

++ I

+++

Storer

I y +

et 01. /Mutation

I +++

Resrarch

,‘1 ++ yy++ I I++

368

~19961

59-101

h I y it++++

+++ I

I

I

I

I

I

I

I

I

I

79

- _- I

I

+ i

I I I .-.vv \ 1. -1 ri’l,

+ 7 /

-t

0.01 0.03 0.10 0.30 1.00 0.000 I o.oocl3 0.0010 o.tMno 0.0 IO0

1% Water 0014

.i’/T Water 0 003

I ‘7c DMSO 0.014

0.10 0.25 0.50 0.75 I .oo

I ‘% Water 0 007

0.03 0.0s 0.07 0.10

0.9

0. I 0,s 0.5 0.7

0.003 0.010 0.030 0.100 0.300

I ‘7c Wnta 0.0 I7

Tetranitromethane [SOS- I J-81 (SA+,Cabs+,SCE+)

t)

0.00 I 0.003 0.010 0.030

I’S I)MSO 0.OO.i

I ‘% 20 mM c1tratc 0 007

d~chromatr 12-O]

N-oxide

Streptorocir1 [18883-66-41

Sodium [77X9-

P-Propiolactone [s7ms7m8] (SA+.Cub\+.SCE+)

-I-Nmwquinolinc [s6ms7ms3 (SA+,ML+,Cahst.S(‘E

99 95 86 79

28 20

020~ 0. I7 I 0.002 0.008 0.018 0.007

104 36 70

104 10s IO2 IO1 I02

I19 91 42 IO I6

Y9 98 I I3 160 194

IO0 I03 IO1 I0 I ND

103 99 88 9

0. I96 0.165 0.141

0.00 I o.oocl 0.002 0.018 0.075

0.213 0.424 0.41 I 0.274 0.258

0.011 0.037 0.098 0.267 0.423

- 0.002 0.001 0.004 0.00x ND

0.000 0.002 0.014 0294

101 IO1 Y9 98

95 IO0 I01 100 I02

IO3 IO4 IO1 IO? 99

100 91 97 IO I I04

99 94 97 9s 96

100 ND

99 IO? IO I

IO4 100 91 6’)

m/(t) -A+)

++ t+ I I ++

L

t/c-j -

+

-/(+) +

-/C-t)

t/C-)

-

( ) (-) (I)

(-)

9s Y7 93 97

08 ‘)I

93 93 IO2

I OS IO3 I04 94 97

98 IO3 III I IO IO9

loo loo 99 99 9s

Y7 98 93 9s 101

I oi YI YI 91

88 84 78 80

96 YO

IO1 96 IO0

88 xx

101 10s IO3

YO 92 100 IO0 83

IO3 103 96 10s 99

95 97 YS 91 98

OS I08 YY 108

I02 I I9 I0 I I02

70 66 97 OS YS

72 92 99 65 62

93 74 60 52 43

107 II3 II4 106 IO1

IO1 III

102 I IS III

II9 YX 81 IO7

I02 IO1 IO2 106

96 96 100 YS 93

Y7 I03 97 65 47

51 38 I’) 9 6

109 Y6 106 104 96

96 IOU 84 86 99

91 90 xx

Iox

93 99 96 96

x5 84 96 83 91

91 98 97 91 84

I02 10s 76 68 64

lr33 107 100 106 91

105 IO1 96 98 9s

II2 I02 107 IO8

~ ~ ~

~~ ~ ~

~ -

~ ~ + t

~ ~ -

-

~ -

~ -

3 (continuctl)

SCE+)

phosphate

results

”

h

1% DMSO 0.018

I % DMSO 0.004

elution slope

Vehicle/

0.002 0.012 0.030

0.001

I .oo 3 7 IO

0.003 0.005 0.003

Induced elution slope ‘

0.03 0.10 0.30

Dose (mM)

99 100 IO1

101 102 I02 101

yIntercept ” ‘7r of control

99 99 97

IO2 102 102 IO1

Fraction DNA remaining after lysis and rinse ’ % of control

-

-

-

DNA Double-strand breaks ’

97 IO2 100

102 102 IO3 101

Cytotoxicity TBDE-0

1OS IO? 102

I04 97 103 97

88 82 78

99 II5 98 I 00

(9 of control) Y TBDE-3 MTT

IO7 106 II2

107 I37 93 I03

ATP

95 106 98

92 92 93 9s

K‘

~ -

~ -

Cdl hlehhing

’ NTP in vitro genetic toxicology tests with positive ( + ). weak positive ( + W). or inconclusive (‘?) results: SA. SalmonellaML. mouse lymphoma, Cabs, chromosome aberrationsSCE. sister chromatid exchange. h The negative (vehicle) control, its final concentration in the media, and the elution slope for the negative control are listed. ‘ Treatment slope minus negative (vehicle) control slope. (P). precipitate/insoluble dose level. ND, not done/no data. ‘t The y-intercept is the intercept (on the v-axis of a semi-log plot of fraction DNA remaining on the filter vs. time) of the linear extrapolation of the 3. to 9-h elutton Ieat-squares line of best fit. Control values ranged from 0.708 to 0.878. ’ Fraction of total DNA remaining after the neutral lysis and rinse steps. a measure of DNA degradation (double-strand breaks). Control values ranged from 0.739 to 0.889. ’ DNA double-strand breaks (DSBs): I-h PACE (Programmed Autonomously Controled Electrodes) pulsed-field gel electrophoresis assay for DNA fragmentation. + , DNA DSBs less than that produced by 40 Gy of gamma radiation. t +, DNA DSBs equal to or greater than that produced by 40 Gy of gamma radiation. Scores determined by densitometric analysis or (for scores in parentheses) by visual examination of ethidium bromide stained gels. Data are considered marginal or equivocal where both visual and densitometric scores ure listed for a particular dose level. B Cytotoxicity assays: TBDE. ttypan blue dye exclusion measured immediately after the 3-h treatment (TBDE-0) or after a 3-h recovery incubation in medium without compound (TBDE-3); MTT. thiazoyl blue dye reduction; ATP, mtracellulur adenosine triphosphate contentK+. intracellular potassium content TBD. to be determined.

Trtmethyl [Sl2-56-11 (SA+,Cabs+,

Compound [CAS No.] NTP genetox.

rahle

M

The the the

results

in Table

compounds induced

excessive presence

slope of’ drug

effects creases

precipitate.

the two cadmium

(XS’k

not

not

metal salts tested. sulfate. did show

confounded

in

of-‘ these confounded

dose elution

J c)t’

by the

in

appeared

by

surcd

increase with

Rcpreacntativc

effects threshold induced

28/33

associated

for compounds 3. Thus. detection

is generally at the in the

was and/or

DNA-damaging

cinogens

that

a dose-related

which

cytotoxicity

of the elution data are included in Fig. the

3 show

produced

by

The

graphs

product

genotoxic

ously

car-

cytotoxic

which give The result\

and

33

been

in

the

(Sina alkaline

and

amycin

benzene.

cells

be

et al.. rlution

(data

in

did but

Diethylnitrosamine

0.01

001 0

3

6

9

Cadmium Sulfate

0

3

Elution

6

Time

(Hours)

0

3

6

Dimethylbenz[A]

9

0

3

Elutlon

Anthracene

6

Time

9

(Hours)

9

previ-

that

rat

positive

LIZ10 find

response

shown)

not were

in

strongly

mouse

We

positive not

has positive

and

assay

did assay

benzo(a)pyrene

weakly

1983)

l%l).

;I weak

at -3 PM

Camptothecin

to

Bradley.

gives

which in the

Adriamycin

reported

there

as mea-

assays.

tested response

aLobenzene.

but

viability

TBDE-3

compounds poaitivc

assays

on cell

4.-l’-methylcnedianiline.

(Ross

wdium dichromate and some rvidencc ot’ loxicity

potassium

the TBDE-O

hrpatocytcs infor

and/or effect

a clear

adriamycin. and

ATP

to be little 5 of

thi\ category in the assay ot by

levels slope.

the

MTT.

that

cells adri-

in

LIZ10

the

elution

slopes decreased with dose as concentrations were increased from 3 to 30 FM. In the studies reported here with mt hepatocytes, there were small increases (O.OOS-0.010) in the induced elution slope in one assay that were not dose-related and were not seen in a repeat assay in the same dose range. The DNA fragmentation assay for DNA double-strand breaks was. however, clearly positive over the dose range from 0.3 to 100 PM but curiously showed an inverse, dose-related effect (decreasing densitometric signal for the amount of DNA entering the gel with increasing dose) in the dose range from 3 to 300 FM. The fraction of DNA recovered in the lysis rinse fraction also decreased with dose from 3 to 30 PM and slow column loading was noted at 30 PM. with complete column clogging at 100 and 300 PM. These observations suggest two explanations for the lack of activity of adriamycin in the assay; (I) that a drug precipitate is interfering with cell lysis and

03

BLEO

DNA mobility and/or (2) that DNA interstrand cross-linking is occurring. Since adriamycin is not known to cross-link DNA. the former explanation seems rnore likely. Azobenzene produced dose-related increases in the induced elution slope but the response was accompanied by extensive DNA fragmentation even at the lowest dose levels tested as judged by the PFGE results and the data for the x-intercepts and fraction DNA eluted in the lysis/rinse fraction. Since the TBDE viability assays, the observation of cell blebbing, and the ATP and K- measurements also indicated dose-related increases in cytotoxicity which were extreme at the highest 2 dose levels tested, the increases in the induced elution slope appear to be associated with a cytotoxic effect rather than a genotoxic effect of azobenzene. Similarly, the increase in the induced elution slope produced by the top dose levjel of 4.4’-methylenedianiline was clearly associ-

STZ

A % N-OH-AAF ~r----l5-----l~-si5

%

DMS

z?

EMS

$2

B Fig. 1. Assay for hepatocyte\ wzre

DNA fragmentation (DNA treated with the indicated

level\ for each compound N-hydrox~-Z-acet~latnlnotluorene

(xc

DSB) by I h PACE/PFGE compounds for 3 h or with

Table 3) mcrcwz from (N-OH-AAF). dimethyl

of \everal genotoxina 40 Gy gamma irradiation

left to right for (A) bleomycin (BLEO) sult’ate (DMS) and ethylmethanewlt’unate

and/or genotoxic carcinogen\. on ice immediately prior and atrcptozottrin (EMS).

(STZ)

Primary rat to ashay. Dose and

for

(B)

ated with cytotoxicity as judged by the precipitous drops in cellular ATP and K- levels, the decline in viability seen in the TBDE assays. cell blebbing and the DNA fragmentation seen by PFGE. Benzene was clearly negative and did not produce any evidence of either genotoxicity or cytotoxicity (which may be due to the volatility of the compound). Benzo[ m]pyrene produced only weak increases in the induced elution slope (0.001-0.010) at soluble dose levels with little evidence of a cytotoxic effect other than a weak response in the 1-h PACE assay for DNA double-strand breaks and cell blebbing at dose levels where compound precipitate was observed. This compound is detected as positive in in vitro unscheduled DNA synthesis (UDS) assays in male Spraque-Dawley rat hepatocytes at dose levels as low as 10 PM after 18-20 h exposures (Selden et al.. 1994). We have found (data not shown) that benzo[a]pyrene is clearly positive in the in vitro alkaline elution/rat hepatocyte assay when using either (I ) hepatocytes from rats induced with phenobarbital and P-naphthotlavone or (2) supplemental inclusion of a liver post-mitochondrial supematant (S-9 mix) from phenobarbital/P-naphthoflavone induced rats. Thus. the negative results in the studies presented here appear to be due to low constitutive levels of metabolic activation in uninduced Sprague Dawley rat hepatocytes which are not sufficient to produce enough reactive metabolites and DNA adducts to produce a positive response in the assay after a 3-h exposure. Benzo[N]pyrene may be somewhat unique in this regard since metabolism to its ultimate electrophile, the dihydrodiol epoxide, requires a 3-step bioactivation sequence involving cytochrome ~450s and epoxide hydrolase (Cooper et al.. 1983). Furthermore, the constitutive levels of the main cytochrome ~450 responsible for benzo[ alpyrene oxidation, CypIA 1, and its associated ethoxyresorufin-O-deethylase activity appear to

Fig. 5. Three-dimensional

graphtcal

representation

of concurrent

be very low in uninduced rat liver as compared to levels in induced rats (Hall et al., 1989: Todorovic et al.. 1991: Van Birgelen et al.. 1995). The pulsed-field gel electrophoresis assays showed that 18/33 of the genotoxic compounds tested produced DNA double-strand breaks in the assay at at least one dose level. Representative photographs of the I-h PACE/PFGE gels are included in Fig. 4. For adriamycin. bleomycin and etoposide. the finding of double-strand break induction is an expected result consistent with the known genotoxic effects of these agents (for a review. see Elia et al., 1991). For several other compounds, most notably dimethylsulN-hydroxy-2-acetylaminofate. epichlorohydrin. fluorene. N-methyl-N’-nitro-N-nitrosoguanidine. 4nitroquinoline N-oxide and streptozocin. a positive signal in the l-h PACE assay was seen only at one or more of the highest dose levels which produced evidence of extensive DNA strand breaks. These effects may reflect double-strand breaks induced by DNA repair processes. such as overlapping sites of DNA excision repair. or they may reflect the early stages of activation of endonucleases in an apoptotic (or necrotic) pathway to cell death. DNA damage. and specifically DNA strand breaks, have been shown (Lowe et al., 1993) to activate genes such as pS3 which modulate the activity of the apoptosis regulatory gene BCL-2 (Miyashita et al.. 1994) and the cell cycle regulatory gene WAF- I /Cip- I /p2 I (El-Deiry et al.. 1994). The size of the DNA fragments detected in the l-h PACE assay ( - SO- 1000 kb) is also not inconsistent with apoptosis induction, since the early stages of endonuclease activation in apoptosis have been shown to involve an initial degradation of the nuclear DNA to - 300 kb and then - 50 kb fragments prior to further degradation, in some cell types, to oligonucleosomal ‘ladder’ sized fragments (Walker et al., 1993). Finally. while cytotoxicity leading to cell necrosis may explain the DNA dou-

utlliL;Ltlon

of induced

elution

slope

and cytotoxtctty

limit

criteria

in scoring

01‘ positive results. Data points t’or all soluble dose level5 of all compound\ tested are plotted. A compound is judged positibc in the in vitro alkalme elution/rat hepatocyte asay if effects at any soluble dose le\cl tested hhow an induced elution slope 2 0.020 with > 70% relative cell viability by the TBDE-3 ‘delayed‘ toxicity assay and ATP content/culture of 2 50% of control values; i.e.. if at least one data point None of the fall5 within the framed box. (A) Rswlth for 18 non-carcinogenic compound\ with no or limited evidence of genotoxicity. compound5 carcinogenh.

tested

(see

For 2X/33

Table

I) met these

compounds

twted

criteria (see Table

for

;I po\itiw 3). thehe

result critt-ria

(False

positives:

for a positive

were

0 % ). (B) met (false

Rewlts negatives:

I’or 33 genotoxins/genotoxic .5/M

or

Ii%).

0.7

Induced Elution

0.5

Slope

0.3

0.1 .o:2Oc; I

-0.1

)E-3(%) ATP (%)

Genotoxic Carcinogens 0.7

Induced Eiution

0.5

Slope

0.3

0.1

0.0~204-4 -0.1

E-3 (%) ATP

(%j

blc-strand breaks seen with cadmium. sodium dichromate and hydrazine. other mechanism< such as localized production of oxygen radicals (Ward et al.. 198.5) or cytotoxic effects due the physical properties of drug precipitate in the case of benzo[ Ll]pyrene and 2.4diaminoanisole sulfate. may be contributing to the positive responses seen in the l-h PACE assay.

The alkaline elution. pulsed-field gel electrophoreais and cytotoxicity data in Tables I and 3 for 61 compounds provide a comprehensive set of validation data which allow for an empirical determination of the appropriate criteria for scoring the in vitro alkaline elution/rat hepatocyte assay. Since cytotoxicity can clearly result in substantialincreases in elution rates due to DNA degradation in dead and/or dying cells. criteria for scoring the assay must by necessity, as previously shown by Sina et al. ( 1983). include cytotoxicity limits which establish the extent of cytotoxicity which can be tolerated in the assay without confounding interpretation of the clution slope increasesand producing false-positive results. The criteria for scoring the assay must also have a minimum induced slope criterion which alows for the small increasesin the 3- to 9-h elution slope which often occur when a small (+ 10-30’5) but significant fraction of the cells in the treated cell population are dead or dying due to compound-in-

duced cytoxicity. For this reason. a criterion based on statistical tests of differences between elution rates for negative control and treatment groups is not appropriate for an assayof this type. With theseprinciples in mind, analysis of the data sets in Tables 1 and 3 indicate that the following criteria result in excellent overall accuracy/predictivity of the assayin correctly classifying compounds as to their genotoxic potential: a minimum induced slope of 0.020. with relative cell viability by the TBDE-3 assay 2 70% and relative ATP content 2 50%. As shown graphically in Fig. 5b. a set of induced elution slope, TBDE-3. and ATP data at any single dose level of a test compound which fulflls these criteria will result in the compound being scoredas positive. Conversely. for a compound to be scoredasnegative, all setsof induced slope. TBDE-3, and ATP data for all dose levels tested must fall outside of these criteria (Fig. Sa). The decision to base these criteria on only 2 of the cytotoxicity assaysevaluated reflects the sensitivity of these assays and their apparent complementarity in detecting degreesof cytotoxic cell damage which are associated with significant increasesin the elution slope. For example, the cytotoxicity of p-nitrophenol and phenformin-HCI is not detected by the TBDE-3 assay at dose levels producing severe ATP depletion and clear increases in the induced elution slope whereasfor chlorpheniramine maleatethe ATP assay is insensitive relative to the TBDE-3 assay at dose3 where DNA double-strand breakageis significant.

Tahlc 4 F,d\e-paitivc

of fenotoxicity.

3.4. Srlrctinll of c,riteriu ,fiw determir~uticm of pwoto.ric qffrcts of test cwnpow~ds in the assa!

u\inf

old

results (fold-mcreaae)

with

non-carcinogen\ v\. new

(induced

with slope)

no or hmited

evidence

Old criteria ” 2 3.0.fold increase in clution \lope 2 70% relative viability by TBDE-0

Compounds

givlnp

falspositlxc

results:

Sodium Ditiocarh rate:

‘I Sma et al..

1983.

of current

assay

New criteria Induced elution 2 70 ‘4 relative and

2 HI”1

data

\et for 28 compounds

0/2x

(O’i

makate

dodecyl sodium

Menthol 7/x+ (75q

)

hlope 2 0.020 viability by TBDE-3

of control

2.4.Dichlorophenol Phenformin Chlorpheniraminc Benryl alcohol

Fillqx)\itivr

Evaluation

criteria

sulfate

I

ATP

content

assay

R.D. Storer Table 5 False-negative new (induced

results with genotoxins/genotoxic slope) criteria

False-negative

giving

false-negative

rate:

carcinogens:

Resrurch

evaluation

results:

87

368 (19961 59- 101

of current

Old criteria a 2 3.0.fold increase in elution slope 2 70% relative viability by TBDE-0

Elution slope criteria for positive: Cytotoxicity assays and limits:

Compounds

rt 01. /Mutation

Adriamycin Benzene 4.1’-Methylenedianiline Trimethylphosphate

data set for 33 compounds

assay

using old (fold-increase)

New criteria Induced elution slope z 0.020 2 70% relative viability by TBDE-3 and 2 50% of control ATP content

VS.

assay

Adriamycin Benzene 4.4’.Methylenedianiline Azobenzene Benzo[ a]pyrene 5/33 (IS%)

Benzo[ alpyrene 5/33 (l57cc)

’ Sina et al., 1983.

An evaluation of the accuracy and predictivity of the assay when applying these criteria to the data set in Tables 1 and 3 and a breakdown of the compounds for which these criteria give false-positive or false-negative results are presented in Tables 4-6. For comparison purposes, we have also evaluated this data set using the original criteria established for the assay by Sina et al. (1983) which utilizes a 3.0-fold increase in the elution slope The results show that the induced slope criteria of 0.020 with cytotoxicity limits based on the TBDE-3 and ATP assays as proposed above gives the highest overall accuracy and predictivity for this set of 61 compounds with 92% of the compounds scored correctly. As shown in Tables 4-6, a reduction in the percentage of compounds giving false-positive results (to OS), by adoption of improved criteria for cytotoxicity. accounts for almost all of the improvement in the

Table 6 Overall accuracy/predictivity of the in vitro alkaline old (fold-increase) vs. new (induced slope) criteria

elution/rat

overall accuracy/predictivity of the assay, with little improvement in the false-negative rate. Reduction in the false-negative rate might be accomplished by utilizing a lower minimum induced slope in the range of 0.010 to 0.012. However, interpolative analysis (between dose levels) of the data for compounds such as phenformin and sodium dodecyl sulfate as well as our experience with the assay in testing proprietary compounds (data not shown) indicate that induced slopes in this range do not provide reliable or convincing evidence of a genotoxic effect of the test compound especially when there is some ancillary evidence of cytotoxicity (for example, the DNA fragmentation, v-intercept drops, and increased amounts of DNA in the lysis/rinse fraction for azobenzene). Furthermore, induced slope increases in this range often do not appear to be dose-related (adriamycin), are not always reproducible

hepntocyte

Elution slope criteria for positive: Cytotoxicity assays and limits:

Past a 2 3.0.fold increase in elution slope 1 70% relative viability by TBDE-0

Compounds with false-positive Compounds with false-negative Overall accuracy/Predictivity

7,‘28 5/33 19/61

a Sina et al., 1983

(25%) (15%) (80%)

assay: evalua!ion

assay

of current

data set for 61 compounds

Proposed Induced elution slope 2 0.020 z 70 % relative viability by TBDE-3 and 2 50 % of control ATP content O/28 5/33 56/61

(0%) (15%;) (92%)

assay

using

(benzo[ alpyrene, adriamycin). and often are associated with dose levels at or near the limit of solubility (benzo[ alpyrene) where precipitate on the elution filters can be problematic. As previously discussed, the v-intercept parameter appears to be, in addition to the TBDE-3 and ATP assays, a useful and reliable indicator of cytotoxicity. Incorporation of a cytotoxicity limit value for this parameter in the criteria for positivity does not appear to be necessary since the percentage of compounds giving false-positive results is reduced to 0% with the TBDE-3 and ATP limits alone. Nevertheless, since the v-intercept can be routinely calculated from the data, we suggest that when borderline positive or negative results are obtained in the assay, reductions in the y-intercept to less than 70% of control values at dose levels where increases in the induced slope are not significant (< 0.020) provide additional supporting evidence that the effects of the test compound are due to cytotoxicity rather than genotoxicity. Where this is the case. our data indicate that the I-h PACE/PFGE assay will also confirm that DNA fragmentation (double-strand breakage) is extensive. The I-h PACE/PFGE assay for DNA fragmentation is clearly a sensitive measure of cytotoxic effects in primary rat hepatocyte cultures which can serve to confirm y-intercept effects. The utility of this endpoint in terms of establishing cytotoxicity limits for scoring the assay is somewhat limited. however, by the fact that DNA DSB produced by genotoxic mechanisms will also produce a positive response in this assay and this endpoint is not therefore solely a measure of cytotoxicity. For example. genotoxic compounds such as etoposide. adriamycin and bleomycin are known to produce DNA DSB (see Elia et al., 1991) and did produce a significant response in the l-h PACE assay. Positive results for etoposide and bleomycin were obtained at dose levels that were also positive in alkaline elution indicating that increases in the 3- to 9-h alkaline elution slope values may provide as sensitive or more sensitive detection of DNA DSB than the l-h PACE assay when they are induced randomly in the genome in all of the treated cells by a genotoxic mechanism (DNA DSB are detected in alkaline elution as two single-stand breaks). Nevertheless, PFGE assays such as the 40-h asymmetric field-inversion gel elec-

trophoresis (AFIGE) are more sensitive in detecting very large molecular weight DNA DSB fragments than the l-h PACE assay (Elia et al., 1993). More problematic is the situation presented by compounds such as hydrazine (and adriamycin) which produced positive results in the l-h PACE/PFGE assay at dose levels that were not accompanied by any significant increases in the elution slope (the negative elution results and the unusual inverse-dose response relationships with adriamyin in the elution and l-h PACE/PFGE assays were discussed previously and may represent an uncommon artifact). The data for hydrazine do, however, show slight dose-related increases in the fraction of DNA recovered in the lysis/rinse fraction as well as slight-dose related increases in cytotoxicity as judged by all of the assays/parameters measured with the exception of the TBDE-0 assay. Thus, the I-h PACE/PFGE assay results for this compound may reflect sensitive detection of a cytotoxic effect leading to endonucleolytic DNA degradation in a small fraction of dead and/or dying cells. If this is the case, then an additional problem in utilizing these data as an indicator or criteria for cytotoxicity is the assays’s exceptional sensitivity and the concern that real genotoxic effects would be discounted if a positive result for DNA DSB in the 1 h-PACE assay were included as an additional limit for cytotoxicity. With more precise quantitation of effects in ethidium bromide stained gels (as is achievable with newer scanning instruments which measure fluorescence from ethidium bromide stained gels directly), a numerical limit value indicative of excessive cytotoxicity could also be derived empirically from validation experiments. Nevertheless, we suggest that the utilization of the l-h PACE/PFGE data (as an indicator of cytotoxicity) in scoring the assay be limited to situations where marginal or equivocal results are obtained using the established criteria based on the induced slope and the TBDE-3 and ATP cytotoxicity assays. In this case, a strong signal in the l-h PACE/PFGE assay which is accompanied by clear evidence based on the y-intercept data of extensive DNA degradation in a significant fraction of the treated cells provides further weight to the evidence for a cytotoxic rather than genotoxic action of the test compound. To evaluate further the performance of the assay

graphs shown in Fig. 6. These compounds were selected based on data from the NTP or on published results for acetominophen (Flaks and Flaks, 1983). benzo[ e]pyrene (Roszinsky-Kocher et al., 1979; WHO, 1983: Lubet et al., 1983) and phenanthrene (Scribner, 1973; Buening et al., 1979; Oesch et al., 198 I). As in Table 1, an abbreviation for the NTP genetic toxicology tests reported to be positive is included beneath the compound name and CAS number. Two of the IO compounds tested, roxarsone and sodium fluoride, gave a positive response in the assay with the new criteria, namely an induced slope of 0.020 with 2 70% relative cell viability in the

utilizing our new criteria, we tested an additional 20 compounds which we have classified as follows: (1) 10 compounds with limited or equivocal evidence of carcinogenicity and genotoxicity, (2) five compounds generally recognized as non-genotoxic carcinogens and (3) five cytotoxic compounds whose genotoxic and carcinogenic potential are unknown. 3.5. Testing qf compounds Mtith limited or equilwcal elsidence of genotosici& and carcinogenici~ Results for the 10 compounds with limited or equivocal evidence of carcinogenicity and genotoxicity are listed in Table 7 with representative elution Roxarsone

0 DMSO 0 137.Cs A4mM n 6mM OBmM VlOmM 0

1% 3 Gy

6

hrpatocyte (Fee Table

9

0

3

6

6

3

Hydrochloride

Elut~on Time Fig. 6. Alkaline elution/rat and/or carcinogenic activity

0.1

/

I 3

Diphenhydramine

0

Sodium Fluoride

9

Eugenol

9

(Hours)

assay for DNA htrand break induction 7 for elution slope and cytotoxicit),

3

0

Elution hy reveral data).

6

Time

(Hours)

compounds

with

equivocal

evidence

of genotoxic

I% DMSO 0.004

0.228 0.159

1.7 2.5

5% Water 0.004

Diphenhydramine~HCI [147-24-O] (Cabs + )

0.003 0.004 0.008 0.012

0.014 0.035

0.04 0.05 0. I 0.3 0.7 I .o

0.000 0.00 I 0.002

0.0 I 0.02 0.03

I % DMSO 0.003

2 - Chloroacetophenone [532-27-41 (Cabs + W)

0.038 0.236

0.002 0.002

0.002(P)

0.1s 0.20 0.30

0.002 0.001(P) 0.003(P)

8 4

73 73

9.5 76 70

97

97 Y4

101 YY 97

99 86

99 100

103 I OS I 06 10s

103

93 Y5

90 92

% of control

95 98

7s 59

equivocal doublebreaks

(+) (+I (+ +I (+ +) (+ +) (++)

+/c-j ++ ++ ++

+ + ++

’

of genotoxicity

+/C-J +/c-j

DNA strand

evidence

Fraction DNA remaining after IyGa and rinse ’

with

74 43

102 IOU 97

7x I8

98 94

I03

103 I05 IO.5

IO3

93 8’)

~ 0.003 0.004 - 0.003

92 93

L

x-intercept ’ % of control

for compounds

- 0.005 - 0.004

slope

Induced elution

0.03 0.07 0. IO

0.0 I

3 7 I0

I

Dov.z(mM)

data

0.40 0.50

h

gel electrophoresis

wK~)

A

Bicphcnol [X0-05-7]

I % Acetone 0.009

rlpyrene

DMSO

[ I92 - 97-21 (.?A+, ML+)

Benzo[

I’/ 0.007

slope

pulsed-field

Vehicle/ Elution

and

[ 103.90-21 (Cabs + , SCE + )

No.] result5 Cl

cytotoxicity

Acetaminophen

Compound[CAS NTP genetox.

Table 7 Alkaline elution.

43

67

81 63 I6 0

87 59 7 0

92 86

100 97 88

IO0 93

100 IO3 102 84

74 24

94 97

94 Y7 xx

9.5 IO1

I04 07 IOY 99

TBDE-3

(9

7 3

86 53

ND

ND ND ND

ND ND

III 99 107 102

67 57 3 I

93 103

109 I I9 98 83

I I7

133 33

134 102

93

74

Y4 94

98 I IS

I.56

IS0 13Y

I25 I37

I OS 102

IOX 108

ATP

99 97

e

88 05

MTT

of control)

carcinogcnicity

IO1

91 6.5

99 98

95 96 YS Y2

106

103 IO4 IO1 IO0

TBDE-0

Cytotoxicity

and/or

3

83 65 X-

91 92

99 105 79 58

104

Y

51

76 89

85

I08 Y7

109 II7

x7 89

93 YS

K+

~

-

-

~ ~

-

+

+

+

-

~ ~

~ ~

-

~ ~

“ebbing

Cell

s

2

2 2 s E

: 2 2 : s

2 c 2

3 ?-

3 27

9 P

I% DMSO 0.01 I

0.038 0.0x2 0. I47 0.187 0.000 0.005 0.013 0.05 I 0.07 I

I 3 5 7 IO

0.004 0.002 0.00 I -0.001(P)

0.002 0.00 I 0.001 0.002

~ 0.00 I - 0.003 0.004 0.013 0.028 0. IO9

4 6 8 I0

0.01s 0.030 0.04s O.OhO

0.02 0.04 0.M 0.0x

0.05 0.10 0.25 0.50 I .OO 2.00

4x 87 86 84 83

16 65 47 60

84 80 81 79

94 92 43 93

93 88 72 49 33 S

88 IO0 90 94 96 86 IO0 44 9s 40 77 79 76 79 90 82 II 74 101 93 96 95 100

-/(+) ++ ++ ++ ++ ++

-/c+j -/C-t) --/c+j -/(+I fi ++ ++ ++ ++ ++ ++ ++ (+) (+) (+) (+) (+)

96 9s 9s 94 9x 97 96 94

97 84 42 89 95

IO? 4s 97 99

95 91 RX 86 77 51

103 93 44 IO1 9h

73 67 38 40

x4 70 56 s2

90 94 81 92

103 97 78 70 63 I7

ND ND ND ND ND

52 51 39 34

89 80 70 66

80 46 x0 7.5

102 10s 99 73 75 63

91 78 85 XI 94

66 58 36 23

36 30 2x 26

X3 93 I IO 104

92 46 103 I06 69 3

10.5 92 40 80 98

41 40 98 90

so 46 47 56

93 96 99 9s

99 95 87 81 65 IS

~ ~ + -

+ +

~

~ -

+ + + +

a NTP in vitro genetic toxicology tests with positive ( + ), weak positive (+ W). or inconclusive (‘?) results: SA. Salmonella; ML, mouse lymphoma: Caba, chromosome aherrationa: SCE, sister chromatid exchange. ’ The negative (vehicle) control, its final concentmtion in the media. and the elution slope for the negative control are listed. ’ Treatment slope minus negative (vehicle) control slope. (P). precipitate/insoluble dose level. ” The y-intercept is the intercept (on the y-axis of a semi-log plot of fraction DNA remaining on the filter vs. time) of the linear extrapolation of the 3- to 9-h elution least-squares line of best fit. Control values ranged from 0.702 to 0.905. ’ Fraction of total DNA remaining after the neutral lysis and rinse steps, a measure of DNA degradation (double-strand breaks). Control values ranged from 0.735 to 0.915. ’ DNA double-strand breaks (DSBs): 1 h PACE (Programmed Autonomously Controlled Electrodes) pulsed-field gel electrophoresis assay for DNA fragmentation. +, DNA DSBs less than that produced by 40 Gy of gamma radiation. + t, DNA DSBs equal to or greater than that produced by 40 Gy of gamma radiation. Scores determined by densitometric analysis or (for scores in parentheses) by visual examination of ethidium bromide stained gels. Data are considered marginal or equivocal where both visual and densitometric scores are listed for a particular dose level. g Cytotoxicity assays: TBDE, trypan blue dye exclusion measured immediately after the 3-h treatment (TBDE-0) or after a 3 h recovery incubation in medium without compound (TBDE-3); MTT, thiazoyl blue dye reduction: ATP, intracellular adenosine triphosphate content; K +, intracellular potassium content.

Sodium fluoride [76X I-49-41 (Cabs-/ + , SCE + /-, ML + 1

K”Xill3”llt: [121-19-71 (ML+)

ML+)

I% DMSO 0.004

Rotenone [83-79-41 (SCE+W.

ML+)

I %’ DMSO 0.004

SCE+,

I % DMSO 0.004

Phenanthrcnc [8.5-01-x] (SA+W)

Eugerl”l [97-53-O] (Cah~f,

TBDE-3 assay and 2 50% of control or greater ATP content. Results for roxarsone (an organic arsenical; 4-hydroxy-3-nitrobenezenearsonic acid) showed that the compound produced a positive response at the 4 mM concentration. This dose level showed evidence of significant cytotoxicity in the MTT assay, reductions in ATP content to 66% of control and extensive DNA fragmentation as judged by the I-h PACE/PFGE data. Whereas there was evidence of delayed toxicity based on a comparison of the TBDE-0 and TBDE-3 assay results, it is possible that these positive alkaline elution results at a relatively high concentration may indicate a cytotoxic rather than a genotoxic effect of the compound. Roxarsone is positive in the mouse lymphoma L5 l78Y mutation assay and an equivocal carcinogen in male rats in NTP studies. Sodium fluoride (NaF) produced a positive result at 7 and IO mM. While there was little evidence of cytotoxicity at these dose levels by the TBDE, ATP, MTT and potassium assays, cell blebbing was noted at 5 and 7 mM and a slight increase in DNA fragmentation was observed over the entire dose-range tested. NaF is clastogenic in vitro and possibly also in vivo (for discussion, see Zeiger et al., 1993) and is considered an equivocal carcinogen based on slight increasesin osteosarcomasin male rats (NTP, 1990). For the 8 of IO compoundstested in this category which gave negative results in our alkaline elution assay,3 compounds(acetominophen, benzo[ elpyrene and phenanthrene) showed little, if any, evidence of genotoxic or cytotoxic effects (Table 7). Four of the

remaining 5 compounds, bisphenol A, 2-chloroacetophenone, diphenhydramine-HCl and eugenol, produced dose-related increasesin the induced elution slope but were scored negative becauseof the extent of cytotoxicity. Interestingly, the increasesin the induced elution slope for these 4 compounds were of sufficient magnitude to have resulted in a positive score by the original criteria for scoring the assay(see Tables 4-6) which utilized 70% or greater relative cell viability by the TBDE-0 assay as the cytotoxicity limit. The 5th cytotoxic compound, rotenone, showed no significant increases in the induced elution slope but did produce evidence of significant cytotoxicity by the ATP, potassium and TBDE-3 assays.Finally, all 5 of these compounds which were scored negative in the assaywhile having produced clear evidence of cytotoxicity also produced a significant increase in DNA fragmentation as judged by the I-h PACE/PFGE assay. Representative photographs of the l-h PACE/PFGE gels for several of these compoundsare included in Fig. 7. 3.6. Testing of ‘non-genotoxic’ carcinogens Five recognized rodent carcinogens for which there is little, if any. evidence of a primary genotoxic effect of the parent compound or a metabolite, were also tested in the assay. Results for these compounds. butylated hydroxyanisole, phenobarbital, piperonyl butoxide, sodium saccharin, and Wyeth 14,643. show that none of the 5 compounds pro-

n M

Y

n-ll

BPA

ROTE

% zl ‘2

PHEN

%

6 g

Fig. 7. Assay for DNA fragmentation (DNA DSB) by 1 h PACE/PFGE of several compounds with equivocal evidence of genotoxic and/or carcinogenic activity. Primary rat hepatocytes were treated with the indicated compounds for 3 h or with 40 Gy gamma irradiation on ice immediately prior to assay. Dose levels for each compound (see Table 7) increare from left to right for bisphenol A (BPA), rotenone (ROTE). and phenanthrene (PHEN). Dose level for 2-acetylaminofluorene (2.AAF) control was 5 FM. DNA molecular weight markers (M) are from 48.5 to 8.3 kb.

duced a positive result as judged by our proposed criteria for scoring the assay (Table 8 and Fig. 8). One of the 5 compounds, phenobarbital did, however, produce a very weak positive at the 10.0 mM concentration as judged by the old criteria of a 3.0-fold increase with 2 707~ relative cell viability by the TBDE-0 assay. Of these 5 compounds, only BHA produced significant increases in the induced elution slope but it did so only at extremely cytotoxic dose levels. A ‘parallel drop’ was a prominent feature of the response to BHA at the lowest two dose levels tested. Phenobarbital produced significant toxicity by the ATP assay and cell blebbing at the highest dose level tested, a dose-related decrease

Butylated

in intracellular potassium content and significant increases in DNA fragmentation by the l-h PACE/PFGE assay (see Fig. 9). A similar result was obtained for the peroxisome proliferator WY 14,643 although intracellular potassium content did not appear to be markedly affected. A slight dose-related ‘parallel drop’ response, as discussed above, was also evident for WY 14,643 and phenobarbital (Fig. 8). Piperonyl butoxide produced some evidence of cytotoxicity evident as cell blebbing and dose-related effects on MTT dye reduction and viability by TBDE. This compound is a potent inhibitor of cytochrome P450 activity (Kamienski and Murphy, 1971) which, although originally found to be non-

Phenobarbital

Hydroxyanisole

s

3

Wyeth

Sodium Saccharin

I

Fig. 8. Alkaline elution/rat elution slope and cytotoxicity

hepatocyte data).

-1

I

6

Elution

14,643

Time

ashay for DNA

0

Elution

(Hours) strand break

3

Induction

by several

6

Time

9

(Hours)

‘non-genotoxic’

carcinogens

(see Table

8 for

1% DMSO 0.01 I

slope

I % DMSO 0.003

Wyeth 14,643 [50X92-23-4]

I %x DMSO 0.006

2% Water 0.007

butoxide

Sodium saccharin [ 128-44-91

Piperonyl [5 I-03-61 (ML+)

0.003

+ )

Vehicle/ Elution

[50-06-61 (SA + W, Cabs?,

ML

” ’

gel electrophoresis,

1% DMSO

16-51

hydroxyanisole

No.] Results

pulsed-field

Phenobarbital

[25013-

Butylated

Compound[CAS NTP Genetox.

Table 8 Alkaline clution,

-0.003 -0.001 0.000 0.002 0.003 0.001

0.75 I.00

82 82

104 I03 94 90

I03 97

0.009 0.012

103 97 88 94

98

82

99 94

99

25

70 19

xx

y-intercept ’ % of control

for non-genotoxic

96 97 I00

data

0.003 0.008 0.0 I3

0.05 0.10 0.25 0.50

I.0 3.0 6.0 IO.0

0.3

-0.001 -0.002 0.001 o.wn 0.004(P)

0.025

0.007

IO

c

0.050 0.075 0.100 0. I so

0.000 0.002

0.000

0.560

0.55 I

0.003 0.126

0.003

0.35 0.45

0.25

elution slope

Induced

cytotoxicity

3 7

9

Dose(mM)

and DNA after

29

++ ++ ++

91 84 85

93 89 84 87 7x

88

98

8.5 84 93

90 99

78

79

86 84

96

91

92 93

103

IO0 91 93 84

98

82 88 87

(6) (-) (-) p/C+) p/C+) -/(+)

95 93

(G) C-J

I03 I03 9s

100 97

99 100 100

-

88

87

-

107

x9

99 94

100

ND

59 0

94 97

103

II6 108 103 107

I05

62 h3 46

69 73

52

h0

56 4x

60

97

107 I I6

I00

II

78

II8 72

MT-l-

(% of control) TBDE-3

98 86

TBDE-0

Cytotoxicity

92

’

99 88

++

++ ++

-/(+)

++

++ ++

+

doublebreaks

101 98

’

DNA strand

-

98 Ion

101

101 108

I00

94

14 56

90

lysis and rinse Q of control

Fraction remaining

carcinogens e

99

49

100 96 68 61

104

75 77 74

78 79

99

II6

II2 I IO

II7

40

II9 98

I

8

loo 75

ATP

90 90

98 92 97 IO1

77 Xh 79

76 X6

II0

100

II2 IO4

104

69

86 57

92

4-

20

78 61

K’

+ +

~ ~ +

-

~ ~

+

+

+ +

+

+

-

-

+

+ +

Cell blebhing

\

? s

‘2 a 2 s c

2 5 Y “s

? ii. s

ic, 9 2 2 e

P

R.D.

Storrr

Fig. 9. Assay for DNA fragmentation (DNA DSB) treated with the indicated compounds for 3 h or compound (see Table 8) increase from left to right (BHA). DNA molecular weight markers (M) are

et al. / Mutution

Re.wurch

368

(I 9961

5%

101

95

by 1 h PACE/PFGE of several non-genotoxic carcinogens. Primary rat hepatocytes were with 40 Gy gamma irradiation on ice immediately prior to assay. Dose levels for each for (A) phenobarbital (PB) and Wyeth 13.643 (WY) and for (B) butylated hydroxyanisole from 48.5 to 8.3 kb.

carcinogenic by the NTP. has more recently been reported to be a rat liver carcinogen when tested at higher dose levels (Takahashi et al., 1994). Finally, the negative results for sodium saccharin were characterized by slight but not significant increases in the induced elution slope and in cytotoxicity which were not markedly dose-related and which were not accompanied by any indication of cell blebbing or DNA fragmentation. The MTT results did, however, show some evidence of significant cytotoxicity at the highest dose-level tested. Saccharin had previously been reported to be positive in the alkaline elution/rat hepatocyte assay by Sina et al. (1983) but our present results do not indicate any genotoxic effects of this compound in the assay using our current test protocol.

3.7. Testing of cytotoxic compounds genotoxicity and carcinogenici~

of unknown

To investigate further the potential for cytotoxicity to confound interpretation of increases in the induced elution slope, we have tested an additional five cytotoxic compounds for which there is little if any information on genotoxicity or carcinogenicity but some knowledge of the mechanism of cytotoxicity. Since some of the compounds we have tested which deplete cellular ATP levels show delayed, as opposed to immediate. effects on cell viability, three of the 5 compounds were selected based on their known abilities to deplete cellular ATP levels by different mechanisms. 2,4-Dinitrophenol, like many halogenated phenol and nitrophenol pesticides, is an

Notes to Table 8: * NTP in vitro genetic toxicology tests with positive (+ ). weak positive f + WI. or inconclusive (?) results: SA, Salmonella: ML. mouse lymphoma; Cabs, chromosome aberrations; SCE, sister chromatid exchange. ’ The negative (vehicle) control, its final concentration in the media. and the elution slope for the negative control are listed. ’ Treatment slope minus negative (vehicle) control slope. (P), precipitate/insoluble dose level. ’ The x-intercept is the intercept (on the y-axis of a semi-log plot of fraction DNA remaining on the filter vs. time) of the linear extrapolation of the 3 to 9 hour elution least squares line of best fit. Control values ranged from 0.702 to 0.905. ’ Fraction of total DNA remaining after the neutral lysis and rinse steps. a measure of DNA degradation (double-strand breaks). Control values ranged from 0.790 to 0.879. ’ DNA double-strand breaks (DSBS): I-h PACE (Programmed Autonomously Controlled Electrodes) pulsed-field gel electrophoresis assay for DNA fragmentation. +, DNA DSBs less than that produced by 40 Gy of gamma radiation. + + DNA DSBs equal to or greater than that produced by 30 Gy of gamma radiation. Scores determined by densitometric analysis or (for scores in parentheses) by visual examination of ethidium bromide stained gels. Data are considered marginal or equivocal where both visual and densitometric scores are listed for a particular dose level. ’ Cytotoxicity assays: TBDE. trypan blue dye exclusion measured immediately after the 3-h treatment (TBDE-0) or after a 3 h recovery incubation in medium without compound (TBDE-3): MTT. thiazoyl blue dye reduction: ATP. intracellular adenosine triphosphate content: K+, intracellular potassium content,

96

R.D.

Storer

et d./Mutcrtim

Research

2,4-Dinitrophenol

368

(1996)

SY-101

Sodium N-Lauroyl

Sulfate

0.01 3

0

“““B

Eii s 0 i! .-m

6

0.010

0

Fig.

IO. Alkaline potential

elution/rat (see

hepatocyte Table

6

3

Elution carcinogenic

9

Sodium lodoacetate

PCltassium Cyanide

1.00

6

9 for elution

assay slope

Time for

9

0

DNA and

strand cytotoxicity

3

Elution

(Hours) break data).

induction

by

6

Time

several

9

(Hours) compounds

of

unknown

genotoxic

and

97

r- 2 c. PI ----- Tc. “,

++++++ ++++++

N

\c c

+++ I

++++

uncoupler of oxidative phosphorylation (Moreland, 1980) which does not produce DNA single-strand or double-strand breaks or DNA protein-cross links in V79 Chinese hamster cells after 30 minute exposures at 5 mM (Shibuya et al., 1991). Potassium cyanide inhibits oxidative phosphorylation by inhibiting electron transport at the cytochrome a-cytochrome a, step (Smith, 1980). Sodium iodoacetate, by contrast, depletes cytoplasmic ATP levels more proximately to the inner surface of the plasma membrane by reacting with a protein sulfhydryl group at the active site of glyceraldehyde-3-phosphate dehydrogenase and thus blocking glycolytic ATP production (Cannon et al., 1991). D-galactosamine, is a potent in vitro and in vivo hepatotoxin by virtue of its effects on aminosugar metabolism leads to accumulation of uridine-5’-diphosphate (UDP) derivatives. impairment of RNA and glycoconjugate synthesis and lethal cell injury (Tran-Thi et al., 1985). The fifth compound in this group is the detergent sodium N-lauroyl sarcosine. None of these 5 compounds produced a positive result by our proposed criteria (Table 9 and Fig. 10). The 3 compounds tested which deplete cellular ATP levels all produced significant increases in both cytotoxicity and in the induced elution slope. The increases in the induced elution slope for the 2 compounds which act by inhibiting mitochondrial oxidative phophorylation were not accompanied by effects on cell viability as measured by either the TBDE-0 and the TBDE-3 assays at the minimum concentra-

tion which gave an induced elution slope of 0.020 or greater. These results further emphasize the importance of including a measure of cellular ATP content as a cytotoxicity limit in the in vitro alkaline/rat hepatocyte assay. As was seen for phenformin, 2,4dichlorophenol and p-nitrophenol (see data in Table I), the sharpest increases in the induced elution slopes occurred when ATP levels were severely depleted (to less than 20-30s of control levels) and DNA fragmentation appeared, especially for sodium iodoacetate (Fig. I I), to be closely correlated with the sharp decreases in cellular ATP content. Finally, a dose-dependent increase in cytotoxicity was seen in the TBDE and potassium assays for sodium iodoacetate and 2.4-dinitrophenol but not for potassium cyanide. The negative results for the detergent sodium lauroyl sarcosine showed a toxicity profile similar to that reported earlier (see Table 1) for sodium docecyl sulfate. D-Galactosamine (GalN) produced little if any evidence of cytotoxicity after a 3-h incubation, in agreement with the results of Tran-Thi et al. (1985) who found significant lactate dehydrogenase leakage was not detectable in GalNtreated hepatocytes in monolayer culture until 26 h after addition of the compound.

4. Concluding

discussion

There are a number of different assays which measure DNA strand breakage in alkali including

Notes to Table 9: a NTP in vitro genetic toxicology tests with positive( + ). weak positive (+ W), or inconclusive (‘?I results: SA. Salmonella; ML, mouse lymphoma; Cabs, chromosome aberrations: SCE, sister chromatid exchange. h The negative (vehicle) control, its final concentration in the media, and the elution slope for the negative control are listed. ’ Treatment slope minus negative (vehicle) control slope. (P). precipitate/insoluble dose level. ’ The ?-intercept is the intercept (on the x-axis of a semi-log plot of fraction DNA remainin, D on the filter vs. time) of the linear extrapolation of the 3- to 9-h elution least-squares line of best tit. Control values ranged from 0.702 to 0.905. ’ Fraction of total DNA remaining after the neutral lysis and rinse steps, a measure of DNA degradation (double-strand breaks). Control values ranged from 0.790 to 0.879. ’ DNA double-strand breaks (DSBs): I-h PACE (Programmed Autonomously Controlled Electrodes) pulsed-field gel electrophoresis assay for DNA fragmentation. + . DNA DSBs less than that produced by 40 Gy of gamma radiation. + + , DNA DSBs equal to or greater than that produced by 40 Gy of gamma radiation. Scores determined by densltometric analysis or (for scores in parentheses) by visual examination of ethidium bromide stained gels. Data are considered marginal or equivocal where both visual and densitometric scores are listed for a particular dose level. ’ Cytotoxicity assays: TBDE. trypan blue dye exclusion measured immediately after the 3-h treatment (TBDE-0) or after a 3-h recovery incubation in medium without compound (TBDE-3): MTT, thiazoyl blue dye reduction; ATP, intracellular adenoqine triphosphate content; K+. intracellular potassium content.

alkaline elution (Kahn and Ewig. 1973). alkaline DNA unwinding (Ahnstrom and Erixon, 1973). alkaline sucrose gradients (McGrath and Williams, 1966), viscoelastometry (Parodi et al., 1981) and the single-cell, alkaline gel electrophoresis (‘comet’) assay (Ostling and Johanson, 1984). Although these methodologies all provide sensitive means of detecting DNA damage induced by genotoxic agents from a variety of chemical classes, alkaline strand break assays have not been widely utilized in in vitro genetic toxicology test batteries. One reason for this may be that in vitro genetic toxicology testing guidelines require testing at high concentrations of test compounds up to and including cytotoxic concentrations. Since extensive endonucleolytic degradation of nuclear DNA can occur very rapidly in dead and dying cells, strand break assays have the potential to be easily confounded by cytotoxic effects of the test compounds. We have found that the problems which cytotoxicity presents for in vitro strand break assays are surmountable if sufficient effort is devoted to developing more sophisticated means of assessing cytotoxic/cytolethal effects and their contribution, if any, to the DNA strand break endpoint being used as an indicator of genotoxicity. The alkaline elution assay has the advantage that the elution data can supply important clues as to the mechanism by which strand breaks are induced, through careful assessment of the kinetics of DNA elution from the filter. Thus, important information can be gained from measuring the amount of DNA eluted in the neutral lysis/rinse fraction (Zwelling et al., 1991; Kaeck et al., 1993: Elia et al., 1993) and our data show the excellent correlation of the v-intercept parameter with toxicity for many cytotoxic compounds. In addition. supplemental use of pulsed-field and conventional agarose gel electrophoresis assays for DNA fragmentation, as shown here and by others (Elia et al., 1993. 1994: Zwelling et al., 199 1; Walker et al., 1993) allows for a more complete characterization of the size range of degraded DNA and can further help to identify and differentiate between apoptotic and necrotic mechanisms of cytotoxicity. The high overall accuracy and predictivity of the assay using the scoring criteria proposed here shows that an in vitro alkaline elution assay using primary rat hepatocyte cultures is a sensitive and reliable

genetic toxicology screening test. The test system is also very rapid. We can routinely test 3 compounds in duplicate at 4-5 dose levels in one 3-day assay, obtaining results that are highly reproducible from assay to assay. Like the unscheduled DNA synthesis assay, the alkaline elution/rat hepatocyte assay has the advantage of measuring primary DNA damage in a metabolically competent target cell. In addition, the test uses primary (normal) rat cells and not an immortalized or fully transformed cell line. Our aim was to improve methods and criteria for the assessment of cytotoxicity in the assay to reduce the percentage of compounds giving false-positive results. We achieved this but with little accompanying improvement in the false-negative rate. To a certain extent, the false-negative rate probably reflects the intrinsic inability of the assay to sensitively detect the genotoxic potential of a few classes of genotoxic compounds due to problems associated with insolubility (e.g., polycyclic aromatic hydrocarbons). volatility (benzene), the nature of the damage produced and/or a low rate of metabolic activation in uninduced rat hepatocytes. These same properties make a number of these compounds difficult to detect in other genetic toxicology assays as well. In attempt to reduce the false-negative rate for the assay, we are currently investigating whether the overall accuracy and predictivity of the in vitro alkaline elution/rat hepatocyte assay can be further improved by using both uninduced and induced rats. As recently shown by others (Shaddock et al.. 1989; Shaddock et al., 19901, utilization of induced rats in a DNA damage and repair assay (unscheduled DNA synthesis, UDS) in rat hepatocytes can improve the ability of the assay to detect the genotoxicity of certain compounds. The criteria we have chosen to use may not be appropriate for other laboratories that use different alkaline elution solutions at different pH values, different elution flow rates, different target cells. and/or other cytotoxicity endpoints. In addition, yintercept effects and DNA double-strand break induction due to endonuclease activation in dead or dying cells may be more or less sensitive as a measure of cytotoxicity depending on the target cell and culture conditions. However, the general principles of and approach to validation that we have taken here are more broadly applicable, e.g.. the concept of

loo

R.D.

Storer et al. / Mutatim

empirically deriving a ‘minimum induced slope or rate’ criterion which does not rely on statistical measures of significance and which utilizes only the elution rate for the terminal phase of alkaline elution should be generally applicable. In addition, the adoption of appropriate cytotoxicity limits determined for multiple, complementary cytotoxicity assays (such as a TBDE delayed toxicity assay together with an assay for cellular ATP content) may have value for in vitro alkaline elution assays using other cell types as well as for other assays which measure DNA strand breaks either directly (alkaline DNA unwinding and the ‘Comet’ assay) or indirectly (SCEs, chromosome aberrations and mouse lymphoma L.5 178Y assays). In vitro DNA strand break assays should therefore, in our view. be considered as highly susceptible to the confounding effects of cytotoxicity unless adequate validation studies have been performed which document the effectiveness of the criteria set forth for the assay in discriminating between genotoxins and non-genotoxic (but cytotoxic) compounds.

Acknowledgements The authors wish to thank Sheila M. Galloway for her critical review of the manuscript and Ken Bamhardt for technical assistance.

References Ahnstrom. G. and K. Erixon (1973) Radiation induced strand breakage in DNA from mammalian cells. Strand separation in alkaline solution. Int. J. Radiat. Biol.. 23. 2X5-209. Armstrong. M.J.. CL. Bean and S.M. Galloway (1992) A quantitative assessment of the cytotoxicity associated with chromosomal aberration detection in Chinese hamster ovary cells. Mutation Res., 265. 45-60. Bradley. M.O. and J.F. Sina (1984) Methods for detecting carcinogens and mutagens with the alkaline elution/rat hepatocyte assay in: B.J. Kilbey. M. Legator, W. Nichols and C. Ramel (Eds.). Handbook of Mutagenicity Test Procedures, Elsevier. Amsterdam. pp. 71-82. Bradley, M.O., G. Dysart. K. Fitrsimmons. P. Harbach. J. Lewin and G. Wolf (1982) Measurements by filter elution of DNA single- and double-strand breaks in rat hepatocytes: Effects of nitrosamines and gamma irradiation. Cancer Res.. 42, 25922597. Bradley, M.O., V.I. Taylor, M.J. Armhtrong and S.M. Galloway

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