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Genotoxicity of polycyclic aromatic hydrocarbons in escherichia coli PQ37
Rt~vcurch. 27X ( I Yl)l) I -I) ( IS’_’ El.wvirr Science Publishers l3.V. All right\ reserved
Mtrurwr7
MLJTGEN
Olh~-I~IR/Y7/%0S.00
017X
Genotoxicity
of polycyclic aromatic V. Mersch-Sundermann,
lnsriiurr
of Medical
hydrocarbons S. Mochayedi
in Escherichia coli PQ37
and S. Kevekordes
Microbiology und Hygienr, Faculty of Clinical Mrdicine Munnhrim.
Unil yrsiry
D-6X00 Munnheim IF. R. G.) (Received
1 November lYY1))
(Revision received 2 July 1991) (Accepted 9 July 1901)
Keywords: SOS chromotest; Polycyclic aromatic hydrocarbons; Genotoxicity
Summary In the present investigation, 32 pol;cyclic aromatic hydrocarbons (PAHs) were tested for genotoxicity in E. coli PQ37 using the standard tube assay of the SOS chromotest. PAHs such as benzo[glzi]fluoranthene, benzo[j]fIuoranthene, benzo[a]pyrene, chrysene, dibenzo[a,l]pyrene, fluoranthene and triphenylens exhibited high genotoxicity when incubated in the presence of an exogenous metabolic activation mixture. ‘The results were compared to those obtained with the Salmonella/microsome test.
In E. co/i PQ37 the /3-galactosidase gene (lucZ) is placed under control of the s&l gent operon, which belongs to the SOS-repair system ($4 : : 1acZ fusion). Accordingly, upon the induction of DNA damages not only the s&f gene, but also the 1acZ gene, is expressed. Hence, the gene product of lacZ, P-galactosidasc, can be measured by a simple assay and thus provides a measure of genotoxicity. In contrast, to obtain a measure of a generalized bacteriotoxic effect, the alkaline phosphatase activity of E. co/i PO37 can be measured as it is an indicator of general protein syn?hesis (Quillardet et al., 1982a, b, 1985; Quillardet and Hofnung, 1985; Hofnung et al., 1989; Walker, 19841.
Correspondence: Dr. Volker Mrrsch-Sunderman% lnstitut fiir Medizinische Mikrobiologie und Hygiene, Fakultit fir Klinische Medizin Mannheim der Universitlt fach 1001123, D-6800 Mannheim I (F.R.G.).
Heidelberg,
Post-
As part of a continuing characterization of the E. co/i genotoxicity assay (SOS chromotest) (Mersch-Sundermann, 1989a, b), we examined the responses of 32 polycyclic aromatic hydrocarbons (PAHsJ. Materials and methods Chemicals
The 32 PAHs were tested by the standard SOS chromotest with slight modifications. The sources of chemicals are described in Table 1. The PAHs selected for genotoxicity testing are representative of those that are abundant in environmental samples (Brune et al., 1979). For testing the PAHs were dissolved in DMSO up to doses of 39-20000 rig/assay or to the limit of solubility in 10 differcnt dilutions. All chemicals were routinely tested in triplicate in the presence and absence of SO derived from the livers of Aroclor 1354-induced rats.
with
Wc follo\v~d :he procedure recommended by Quillurdct and Hofnung ( 1985) with slight modifica:ions. I ml ctf an E. I-V!! PO37 overnight culture \\;is diluted :;ith 9 ml L-riedium. Then hOl)-~.~l portions of this dilution ; = 5 X lOh cfu) we c distributed into tubes containing 20 +I of gradual dilutions of the PAHs to be tested. Using the text procedure with metabolic activation S ml of a modified S9 mis ~5OYi 52) mix) replaced 9 ml of L-me&m (see below). These mistures were incubated for 2 h at 37 OC
T.ABLE
I
CtlAR;\(‘TERIZ(\TIC)N
OF P.i\t?h
Anthanthrene Xnthracrnr Eirnz[tr]anthraccne Benzo[ h]fluoranthenr Brnzo[ g/n]iluoranthcnr Benzo[ j]tlrwrunthcnc Brnzo[~rjIluorenr Bcn,w[ h]tluorznc Benzo[ ,gIzi]pr+ne Bcn~ti[ cr]p)rrnc‘
32.32 'i7 _. _ 12 2lh.‘S 2lh.2S 77fb.34 75-l _. -..i
Benzo[eJpyrene Chnsrnr Coronrne
3’ -._3 -_ “Y ._‘Y __.
Dihrnz[u.c.];lnthrctcrn~
27s.1 27S.J
Dihrnz[tr.ll]anthracene Diiiazo[ [l.l]pyrenr Di‘nrnzo[ tr.h]pyrrnr Dihenzo[ ~.i]pqrrnr 7. I?-Dimethylhenz[u]anthracrnr
.Wl).3l
32.4 302.4
Jacob Srrw 13675 Srrva l-1530
YY’;
Aldrich .27.533-b
> 995 > YYci YWi YXri > YXri YX’i YYri Y5Ci 97% YX? YV-i
Jacob J;icoh Fluka 11-190 Fluka 124’!5 Aldrich B YOO-Y Serb3 IJxoo Aldrich B1.010-2 Aldrich CX.OOO-X Aldrich CX.GW I Serva 19275 Serva lY7XO
> YYci
Jacob
301.1
> YYci 9X?
Jacob Sigma DO33
2Sh.35
> W’i
Fluka
3.bDimethklphenanthrenr Fluoranthene Fluorrnr Indeno[ 12.3~ctllpyrenr 2-Methylanthracene Y-blcthylanthracenr
Yx’; YSCi
> YYc;
39570
Aldrich 26.190-3 Servu ‘I539 Selva 2155? Jacob
! Y23
Atlanta
I.3533
Atlanta
7121
%Mrthylcholltnthrene
I913 35X.3h
2-Mrthylphcnanthrenr Naphthalenr PU$UW
I’)‘.76 11X.1 157_.__ 3’ _.
Aldrich
Phenanthrene Pyrrnr
17x.1 ‘01.3 ‘3.‘Y
Srrva
Triphrnylrnr
Fluka
hh’30
Pl.l20-4 32005
Serva 33hh5 Aldrich
Metabolic actirution In contrast to the procedure described by Quiilardet and Hofnung (1985) we used S9 mix containing only 50% of the standard 9000 x g supernatant (see also Marzin et al., 1986). The composition of the S9 mix has been described by McrschSundermann et al. (199lb).
7b.11)4-6
Sigma N3XO Aldrich
agitatic>n. To prevent measurement problems due to the turbidity of the S9 mix a short centrifugation step (2500 x g. 5 min) followed. 550 ~1 of the supernatant was decanted carefully and the rcmainir,g bacterial pellets were resuspended in 550 ~1 of a 0.9% NaCl solution. After mixing thoroughly, a further centrifugation step with decantation of 550 ~1 supernatant and resuspension with 550 ~1 0.9% NaCl solution followed (washing step). Then. 300~~1 portions from each tube were drawn and placed in fresh tubes, such that the-.e were 2 equal series of tubes each Tontaining a volume of 300 ~1. To determine the induced P-galactosidase activity, 2.7 ml of bg buffer and 600 ~1 of 0.4% 4-nitrophenyl-fi-o-galactopyranoside !QNPG) solution were added to each tube of one series. The mixtures were incubated for 30 m;n at :‘7 o C in a water bath. The incubation was stopped with 2 ml of I M sodium carbonate. The absorbance at 405 nm was read against a blank containing all ingredients but lacking bacteria. The determination of alkaline phosphatasc activity was similar to the P-galactosidase assay except that ap buffer replaced the bg buffer and a 0.4% 4-nitrophenyl phosphate (PNPP) solution replaced the ONPG solution. The conversion of PNPP was stopped with 2 ml of I.5 N sodium hydroxide. Positive controls, equipments and sources have been described elsewhere (Mersch-Sundermann et al., 1989a, b, 19Yla). The photometer was a Hitachi model 1170-60 digital double-beam spectrophotometer (horizontal beam). Measurements were made with 1 cm standard plastic cuvettes.
TKXO-0
Calculation The P-galactosidase (bg) and al!:sline phosphatase cap) activities were calculated according to the simplified version recommended by Quillardet and Hofnung (1985): units = A405 x
TABLE 7 GENOTOXlCiTY Compound
OF P.Ws
IN Eschrrichiu
Dose
(/.&assay)
coli PO37
(TUBE ASSAY) IN = 3)
Assay ( - S9J
Assay ( + 94)
Pgai
ap
(units)
(units)
0.156 0.625 2.500 10.000
5.25 5.63 5.73 6.18 6.19
Anthracene
0.000 0.156 0.625 2.500 10.000
Benzlalanthracene
IF
SOSIP
17.4; 17.37 17.41 17.29 i7.:5
1.oo 1.08
0
5.19 5.23 5.23 5.23 5.25
0.000 0.156 0.625 2.500 10.000
Penzo[ h]fluoranthene
&gal iunits)
av (&iitsJ
ratio
SOSiP 0.132
I .09 1.19 1.20
9.63 9.51, 8.97 4.84 8.73
1.uu
5.49 h.24 10.37 13.80
1.25 2.13 2.89
16.88 lb.84 16.81 16.76 16.20
1.00 1.01 1.01 1.02 1.05
5.37 5.56 5.91 6.31 6.41
9.33 9.09 8.92 x.77 8.19
1.00 1.06 1.15 1.25 1.36
0.010
4.57 5.01 4.99 5.03 5.31
lb.27 16.13 16.12
5.32 5.49 5.79 6.96 18.91
8.85 8.73 8.73 8.67 8.65
1.00 1.05 1.11 1.34 3.64
O.!%J
16.16
1.00 1.11 1.10 1.10 1.18
0.000 0.156 O.b25 2.500 10.000
5.45 5.48 5.52 5.49 5.15
17.25 17.15 17.19 17.19 16.89
1.oo 1.01 I .02 1.01 0.96
5.49 5.95 6.41 8.12 14.76
9.03 8.95 8.91 X.81 8.69
1.OtJ 1.09 1.18 1.52 2.79
0.045
Benzo[ghi]fiuoranthene
o.oon 0.155 0.625 2.500 10.000
4 Y3 5.08 4.99 5.05 5.09
16.15 16.08 16.16 16.09 16.01
1.oo 1.03 1.01 1.03 1.04
5.40 5.63 6.55 12.51 33.40
9.56 9.41 9.08 X.97 8.84
1SHJ I .06 1.28 2.47 6.h’)
0.340
Benzo[ j]fluoranthene
0.000 0.156 0.625 2.500 10.000
4.72 5.39 5.45 5.41 5.40
16.64 16.56 iii.45 16.56 16.68
1.oo 1.15 1.17 1.16 1.15
5.07 6.00 6.85 15.77 19.15
9.21 8.89 8.76 8.67 8.32
1.00 1.23 1.42 3.31 4.18
0.254
Benzo[ rc]fluorene
0.000 0.156 0.625 2.500 io.c00
4.96 5.40 5.37 5.43 5.48
lb.97 16.49 16.52 16.48 16.49
1.00 1.12 1.11 1.13 1.14
4.97 4.91 4.88 5.00 5.00
X.56 8.56 8.52 8.57 8.59
i .uo 0.99 0.94 1.oo 1.0
0
Benzo[ hlfluorene
0.000 0.156 0.625 2.500 10.000
5.32 5.41 5.41 5.3’) 5.45
17.37 17.35 17.33 17.32 17.29
1SJO 1.02 1sJ2 1.02 1.03
5.04 5.45 5.91 6.80 9.61
8.43 X.28 8.11 7.07 7.ni
1.oo I.10 1.22 1.43 2.tJ6
0.024
Benzo[ ghi]perylene
0.000 0.312 1.250 5.000 10.000
5.33 5.39 5.37 5.43 5.39
17.19 17.15 17.08 17.05 17.13
1.oo 1.01 1.02 1.03 1.01
4.85 5.83 6.05 7.25 9.15
8.85 8.28 8.03 7.84 7.X0
i .OO 1.29 1.38 1.69 2.14
0.033
Anthanthrene
0.000
16.03
5.35
1.04
Campound tnclmc)
Benzo[a]pyrenr
Ben& r]pyrene
Chrysene
DWX (j.lg/assay)
Dibenz[a.c]anthracene
Dibenz[o,/r]anthracene
B-gal (unitsi
0.243
5.02 6.22
9.19 8.99
1.00 1.27 2.17
16.72 15.83
1.oo 1.15
0.625 2.500
5.91 6.17
15.87 15.87
1.30 I .36
10.25 24.91
8.67 8.54
10.000
6.28
15.63
1.41
31.13
8.34
0.000 0.156
5.13 5.17
17.15 17.05
I .oo 1.01
4.79
8.60
0.625 2.500 10.000
5.21 5.25
17.08 17.07
1.02
5.43 5.53
8.40 8.27
5.21
17.07
1.03 1.02
6.63 6.93
8.16 7.91
0.000 0.156 0.625
4.16 4.hO
15.25
1.oo
15.05 15.05
1.12 1.14
5.12 5.88
8.55 8.39
15.04 14.92
1.16 1.17
6.32 12.79
8.27 8.09
35.59
8.00
15.81
1.00 106
5.21
8.97
5.65 5.75
8.85 8.88 8.72
4.68 4.75 4.77
0.000 0.05 I
4.39 4.48
0.206 3.825 3.300
4.49 4.52 4.44
15.31 15.35 15.40 15.36
0.000 0.156 0.625
4.72 4.85 4.88
16.09 16.12
2.500 10.000
4.85 5.56
16.04 15.93
0.000 0.156
4.68 4.84 5.09 5.12 5.15
0.000
4.55
pyrene
0.156 0.625
5.05 5.11
2.500 10.000
4.85 5.01
0.000 0.052 0.206 0.825
5.47 5.47 5.17
3.300 Dibenzo[Q,i]pyrene
P-gal (units)
5.21
Dibenzo[a,i]-
pyrene
SOSlP
4.79
0.625 2.500 10.000
Dibenzo[Q,h]-
ratio
IF
O.OOil 0.156
2.500 10.000 Coronrne
Assay ( + S9)
Assay ( - S9)
0.000 0.156 0.625 2.500 10.000
16.39
1.05 1.06 1.04
6.13 6.85
8.41
1.00 1.16
1.58 1.00 1.17 1.28
1.00 1.10
1.00 1.10
1.05 1.05
6.15
8.73 8.37
1.22 1.74
8.12
2.99 1.oo
5.24 5.55
9.29 9.08
16.20 16.03 15.93
1.09 1.11
5.60 6.76
8.92 8.51
1.13
11.05
8.29
16.43 16.13
1.00
5.12 10.08
1.41
1.oo 2.02
7.85 7.79
12.55
1.00
16.11
1.09
20.49 55.67
16.09
1.13
46.35
5.39
17.33 17.23 17.28
1.oo 1.01
5.38
5.40
5.79
9.80 9.59
1.02
10.54
6.21 6.93
9.37 8.95
1.oo
10.35
8.79
5.04
17.25
8.87
1.00
16.84
1.oo 1.03
5.09
5.07 5.13
8.79 8.77
1.13
1.04
5.68 7.56
1.05 1.09
12.31 18.99
8.75
1.50 2.45
8.35
3.96
5.35
16.76
0.117
1.10 1.21
1.02
16.88 16.81
2.100
4.15
17.27 16.56
5.17
0.039
2.36
9.05
16.13
0.104
1.08 1.11
8.84 n.75
1.13 1.14
0.027
1.11 1.21 1.40
8.97
1.oo 1.04
0.221
2.64 7.43
9.24
16.32 16.25
0.032
1.20 1.46
5.33 5.69
1.21
0.605
5.34 6.84
1.oo 1.05
8.41 14.01
SOSIP
1.41 2.14 0.174
TABLE 2 (continued) Compound (name)
Dose (/Lg/assay)
Assay t - SY)
Assay ( + S9)
P-gal (units)
aP (units)
IF
5.07 5.65 5.71 5.73 6.01
17.17 16.56 15.52 15.33 15.33
1.00 1.16 I .25 I .27 1.33
0
5.29 5.69 6.44 X.51 i5.80
9.07 8.91 X.80 x.53 x.44
1.00 I 0’) 1.25 1.71 3.21
0.072
0.156 0.625 2.500 10.000
3,6-Dimethylphenanthrene
0.000 0.156 0.625 2.500 10.000
4.89 5.16 5.19 4.60 3.84
16.88 16.76 16.83 16.24 15.55
1.00 I .07 1.07 0.98 0.86
0
5.43 5.72 6.47 6.77 7.71
9.55 9.4x 9.47 9.47 9.47
1.00 1.06 1.20 1.26 1.43
0.007
Fluoranthene
0.000 0.156 0.625 2.500 10.000
5.01 5.04 5.07 5.11 5.09
16.84 16.71 15.52 14.11 14.04
1.00 1.02 1.19 1.22 1.22
0
5.32 7.16 10.95 30.32 40.57
9.11 X.91 x.75 8.57 8.24
1.oo
0.4 I3
0.000 0.156 0.625 2.500 10.000
4.79 4.89 4.85 4.63 3.47
17.05 16.08 16.09 14.47 9.25
1.00 1.08 I .08 1.14 1.33
0
5.16 5.16 5.16 5.12 5.11
8.79 8.81 8.60 8.89 8.73
0.98 1.00
Indeno[ 1,2,3-cd]pyrene
0.000 0.156 0.625 2.500 10.000
5.59 5.65 5.75 5.39 5.21
17.31 17.29 17.27 17.09 17.03
1.oo I .02 1.03 0.98 0.95
0
5.15 5.31 5.83 6.75 10.35
9.0 1 8.96 8.93 8.84 x.97
1.00 1.04 1.14 I .34 2.02
0.036
2-Methylanthracene
0.000 0.156 0.625 2.500 10.000
5.11 5.46 5.52 5.52 5.49
17.49 17.37 17.47 17.40 17.41
1.oo 1.08 1.08 1.09 1.08
0
5.11 5.77 6.53 9.01 9.81
9.13 9.04 E.85 8.75 8.33
1.OO 1.14 1.32 1.84 2.11
0.052
9-Methylanthracene
0.000 0.156 0.625 2.500 10.000
5.16 6.20 5.45 2.28 1.23
17.40 17.31 16.08 5.52 2.67
1.oo 1.20 1.14 1.39 1.55
0
5.16 5.60 6.32 10.88 13.87
9.53 9.29 9.16 8.93 8.78
I .oa 1.11 1.27 2.26 2.93
0.102
3-Methylcholanthrene
0.000 0.156 0.625 2.500 10.000
4.80 5.63 5.64 7.44 11.25
17.05 16.32 16.29 16.35 16.03
1.00 1.23 1.23 I .62 2.50
0.036
5.41 5.80 9.25 18.96 22.03
8.92 8.88 x.77 8.69 x.53
I .oo I .08 1.74 3.59 4.25
0.194
2-Methylphenanthrene
0.000 0.156 0.625 2.500 10.000
4.95 6.00 5.83 1.57 1.37
16.47 16.37 15.36 2.92 2.65
1.oo 1.22 1.26 1.79 1.73
0
5.43 6.12 7.28 10.92 9.08
9.39 9.21 9.35 9.25 9.31
1.00 1.15 I .35 2.04 I .6Y
o.ah8
7,12-Dimethylbenz[a]anthracene
Fluorene
0.000
SOSIP
P-gal (units)
aP (units)
ratio
SOSIP
1.38 2.14 b.06 8.43 1.oo
0
I .oo 1.02
Naphthalcne
-l.c)b
:7.1
I .oo
S.-u 5.20
16.7’) I(,.09
I.12 I.12
5.4x 5.4X
5.15 4.h4
I-l35 I I .85
I.20 I.36
5.11
5.20
17.37 17.29 17.11 17.OY
I .oo
s.3 5.29 5.27 S.23 Phrnanthrrnr
0
1)
I .02 I si3
5.01 5.h’)
1.32
lh.33 lb.15
I .oo 1.04 I.10 I.11
-1.89
15.0x IS.09 15.04
5.35 5.48 5.60
I.10
S.Yh
S.2-I _5 ._77
17.21 17.16
I .oo I.01
5.3 I
I7.04 17.13
I .02
h.0’) 7.4x
I.01 I .07
17.79 7’ 9’) __.
Ih.24
0
I .03
1000/r (A405 = optical density at 405 nm; t = substrate conversion time in minutes). The induction factor IF was calculated as the ratio Rx/R,, (R, = bg/ap determined for the test chemical; R,, = bg/ap determined for the negative control). The SOS-inducing potential (SOSIP) that describes a single and comparable parameter for the genotoxic potency of a compound was determined as the steepest slope of the IF dose-response curve (increase of IF per nmole of compound). A compound was classified as ‘non-reactive’ (‘not genotoxic’) if the induction factor remained < 1.5, as ‘marginal’ if the induction factor was between 1.5 and 2.0 and as ‘reactive’ (‘genotoxic’) if the induction factor was in excess of 2.0. Additionally, to avoid false-positive results due to bacteriotoxic effects (decrease of ap activity), a further requirement for genotoxicity was a
0
0.0 I 7
6.71
4.35
5.24 5.29
SOSIP
h.05 6.49
I .03 I .t17
I .03 1.34
-I.% 1.x’) 4.c)Y
ratio
5.43
IS.95 IO.39
4.81
ap (units)
S.48
I .oo
4.28 1.7h
Triphenylene
1h.h’)
(units)
lb.57 1b.M
511 S.23 5.07
Pyrene
I
SOSIP
,fz-gal
3P (units)
IF
(units)
p-g.31
S.!)7 5.31 6.49
0.053
K-3 I%4 0
0
continuous compound
>.73
5.16
0
0.260
increase in bg activity with increasing concentration.
Results and discussion The genotoxic activities of 32 PAHs on E. coli PQ37 are shown in Table 2. Anthracene (SOSIP = 0.01) (Fig. lA), benzo[a]fluorene (SOSIP = 0.0241, coronene (SOSIP = 0.027), 3,6-dimethylphenanthrene (SOSIP = 0.0071, fluorene (SOSIP = 01, naphthalene (SOSIP = O), perylene (SOSIP = 0.017) and pyrene (SOSIP = 0) showed little or no genotoxic activity (IF < 1.5) either in the presence or in the absence of S9. In addition benzo[e]pyrene (IF (max) = 1.9, SOSIP = 0.032) displayed a weak response. All the other compounds induced the SOS system of E. co/i PQ37 in the presence of S9 mix.
7
The highest activity was shown by dibenzo[~~./]pyrcne with a SOSIP = 2.1 (IF tmax) = 12.5) (Fig. 10. In the absence of a metabolizing system only benzo[a]pyrene (SOSIP = 0.243) and 3-methylcholanthrene (SOSIP = 0.036) showed genotoxic effects. The influence of the USCof S9 mix containing less 9000 x g supernatant from Aroclor 1254-induced rat livers than the standard S9 mix on the SOS response of E. co/i PQ37 is shown in Fig. 2. Whereas we had only a very weak or questionable
response testing O-634 ng benzo[alpyrcne/assay using the standard SC)mix (54 ~1 9000 x g supernatant/assay), the response was increased (up to IF = 2.0) (reactive) when using the ‘50% SC)mix (27 ~1 Sc)/assay) and only 312 ng benzo[a]pyrene/assay. Additionally the SOSIP was significantly higher using lower SC)concentrations (data not shown) (Mersch-Sundermann et al., 199lb). The cause is probably the inactivation of trace amounts of /I-galactosidase by components present in the 9000 X g supernatant. The SOS re-
induction factor 3.0
units A
units
Anthracene (+ S9)
15
inductton factor - 3.0
B Anthanthrene
15
(+ S9)
t
I; si
2.5
10 -.
10
0
2.0
. . 2.0
5 .. . . 1.5
1.5
0 .-
1.0 i
I
500
SOSIP = 0.142
. . 1.0
I
:,:~::~:.::~:~::~::‘:::‘!:
0
. . 2.5
t
1000 1500 nglassay
2000
_.0
2500
. 500
1000 1500 nglassay
2000
2500
induction facto! Dlbenzo(a,l)pyrene
I
+ + +
Fig. I. P-Galactwidase (nonreactive
compound),
and ;dkaline
:,
500
:: ::::,z: :::,:: 1000 1500 2000 nglassay
’ 2500
R-galactosidase activity alkaline phosphatase activity induction factor
phosphatnse
(5) anthanthrrnr
‘:;:’
0
(+ S9)
activity.
(reactive
calculated
compound)
induction
factor
and SOS inducing
and (C’) dihenzo[a.l]pyrcne
(extremely
potential
of (A) anthracent:
highly reactive
compound).
x inditctton 5--
factor
but only verified for the testing of PAHs (Mersch-Sundermann et al., 199lb). A comparison of the SOS chromotest results with those obtained in the Salmonella/microsome assay (Sakai et al., 1985) for 18 PAHs showed a good correspondence between genotox-
4-
3-
2r
TABLE
3
GENOTOXICITY
AND
MUTAGENICITY
OF PAHs
i-
Name
i
Q10
20
30
40
90009
supernatant
B(a)P
50
60
70
80
concentration
0.078
llg
4
0.156
ug
-i-
0.625
Ug
-
1.25 “9
-
0,312
-
2.5
tlg
ug
Fip. 2. Influence of the 9000 X !: supernatant concentration on the response of E. colr PQ37 testing hrnzo[a]pyrene.
sponse is further increased when using very low SC) concentrations (6 PI/assay) and a low compound concentration (Fig. 2). It is to be noted that the increase of SOS-inducing activities when using this modified S9 mix is not a general rule,
induction factor
8 testing5000
nella assay a
nglaasay 0 fluoranelerle
Anthanthrene
2.89
Anthracene Benz[a]anthracene
1.36 3.78
Benzo[b]fluoranthene Benzo[ g&]fluoranthene
3.21 8.11
BenzoIjJfluoranthene Benzo[ alfluorene Benzo[ blfluorene
4.44 1.01 2.23 2.14
Benzo[ &:lli]perylene Benzo[ cllpyrene Benzo[ rjpyrene
6.94 I .YO
Chrysene Coronene
7.43 I .47
Dibenz[u.c]anthracene
2.99
Dibenz[a,h]anthracene
2.62 12.55
Dibenzo[ a,l]pyrene Dibenzo[cr,h]pyrene
2.62 4.26
anthracene 3.6-Dimethylphenanthrene
3.66
Fluoranthene Fluorene
x.71
I so I .02
6
Indeno[l,2,3-cdlpyrene
2.59 2.35
5
2-Methylanthracene Y-Methylanthracene
8 Chrysene
2.93 4.84
3-Methylcholanthrene
2.04
2-Methylphenanthrene Naphthalene 7.1%Dimeth benz(a)amhacene e QMet 1cylanthracene Phenanthrene 0 2-Methylanthracene 2-MethylPhenanthrene ti @ Benzo(ghi)perylene
3 2
1.OY
1.46
Perylene Phenanthrene Pyrene Triphenylrne
+ _ + + + + + + + (+) + _
+ - 1) + + + + ? ‘> + + + + + +
+ + + + +
0 + +
+ -
+ _
+ _
+ -
+ + + + + -
+
2.94 I.21
-I-
5.03
+
-
+
+ + + 0 _ + - 1) + +
’ Data I’rom McCann et al. (1975), Brune et al. (1979), Bartsch et al. (1980). Haworth et al. (1983). Sakai et al. (1985).
0 0
100
200
cr::::::::::::::::::::::: 300 400
Brahms et al. (1987).
500
( + ) weak genotoxicity (I.5 < IF > 2.0); 0 data inadequate or
SJyphimurium TA97 revertants per plate Fig.
genotoxicitv
Dibenzo[u.i]pyrene 7.12-Dimethylbenz[a]-
7
Salmo-
IF (max) 90
(@assay)
-
SOS chromotest
3.
Correlation
of genotoxicity
on E
not available; ? conflicting reports; I) negative in TAl535, coli
PQ37
mutagenicity on Sulmonrlla ryphimurium TAY7.
and
TA1537, TA1538, et al.. 1385).
TA98,
TAlOO;
positive only TA97 (Sakai
icity in E. coli PQ37 and mutagenicity in Salmonella typhimurium TA97 (Fig. 3) (Table 3). In view of this and other studies (von der Hude et al., 1988) the SOS chromotest seems to possess some practical advantages over the widely used Salmonella,/microsome assay (Maron and Ames, 1983). The SOS chromotest is easy to perform, the results are obtained within a few hours and only a single bacterial strain is required. Thus, further characterization of the assay with respect to the response of diverse chemical classes, the standardization of the test protocol and the determination of the structural basis of SOS-inducing activity are warranted to evaluate the role of the SOS chromotest as part of a battery for the prediction of carcinogenicity.
Press, London. pp.
Maron. D.M.,
and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test. Mutation Rcs.. 113.175.-21.5. Marzin.D.R., P. Olivier and H. Vophi (1986) Kinetic detcrmination of enzymatic activity and modification of the metabolic activation system in the SOS chromotest. Mutation Res., 164, 353-359. McCann. J.. E. Choi. E. Yamasaki and B.N. Ames (1975) Detection of carcinogens as mutagens in the Salmonella/ microsome test: assays of 300 chemicals. Proc. Natl. Acad. Sci. (U.S.A.), 72, 5135-5139. Mersch-Sundermann.
V., A. Hofmeister.
G. Miiller
and H.
Hof (l989a) Untersuchungen zur Mutagenitat organischer Mikrokontaminationen in der Umwelt. III. Mitteilung: Die Mutagenitat ausgewahlter Herbizide und lnsektiride im H.
Hofmeister
(1989b) Untersuchungen zur Mutagenitat organischer Mikrokontaminationen in der Umwelt. IV. Mitteilung: Die
We thank Herbert S. Rosenkranz from the Department of Environmental and Occupational Health at Pittsburgh, PA for his advice and the reading of our manuscript. Moreover, we thank P. Quillardet and M. Hofnung from the Institut Pasteur, Paris, for the Escherichia coli strain PQ37 and J. Jacob from the Biochemical Department for Environmental Carcinogens, Ahrensburg, for a number of the test compounds. Furthermore, we thank W. Meister for his laboratory help.
Mutagenitiit leichtfliichtiger Organohalogene im SOSChromotest, Zbl. Hyg., 189. 266-271. Mersch-Sundermann. V.. S. Kevekordes and S. Mochayedi (1991a) Sources of variability of the Esrhericl~iu coli PQ37 genotoxicity assay (SOS chromotest). Mutation Res.. 252. 51-60. Mersch-Sundermann. V.. F. Wintermann and S. Kern (199lb) Sources of variability of the SOS chromotest: influence of the metabolizing system on the response oi E. ct,li PQ.77. in press. Quillardet. P.. and M. Hotnung (19X5) the SOS-Chromotest. a calorimetric bacterial assay for genotoxins: procedures, Mutation Res., 147, 65-78. Quillardet. P.. 0. Huisman. R. d’Ari and M. Hofnung (1982a) SOS chromotest. a direct assay of induction of an SOS
References Bartsch. H., C. Malaveille. A.-M. Camus, G. Martel-Planche. G. Brun. A. Hautefeuille, N. Sabadie and A. Barbin (1980) Validation and comparative studies on IX0 chemicals with Salmonel/u r)?phimrtr;um strains and V7Y Chinese hamster cells in the presence of various Mutation Res., 76, l-50.
metabolizing
Brams, A., J.P. Buchet. M.C. Crutzen-Fayt,
systems.
C. de Meester, R.
Lauwerys and A. Leonard (1987) A comparative study. with 40 chemicals, of the efficiency of the Salmonella assay and the SOS chromotest (kit procedure). Toxicol. Lett., 38, 123-133. Brune, H., R. Deutsch-Wentzel,
S. Dohbertin.
G. Grimmer,
M. Habs. J. Jacob J. Misfeld, U. Mohr. G. Obersdoerster. F. Pott, D. Schmaehl, P. Schneider and D. Steinhof (1979) Luftqualitatskriterien fiir ausgewlhlte polyzyklische aromatische Kohlenwasserstoffe, Erich Schmidt, Berlin. S.. L. Lawler.
Genetic Risk Assessment, Academic 325-136.
SOS-Chromutest. Zbl. Hyg., 189, 135146. Mersch-Sundermann, V., G. Miiller and
Acknowledgements
Haworth,
Hofnung. M.. P. Quillardet. 0. Goerhch and E. Touati (19x9) SOS chromotest and the use of hack% for the &tectjon and diagnosis of genotoxic agents. in: bie7.v Trends ;n
K. Mortelsman,
W. Speck and E.
Zeiger (1983) Salmonella mutagenicity test results for 250 chemicals, Environ. Mutagen.. Suppl. I. 3-142.
function in Eschericlri~ co/i K-12 to measure gecotoxicity. Proc. Natl. Acad. Sci. (U.S.A.!, 79, 5971-5975. Quillardet, P., 0. Huisman. R. d’Ari and M. Hofnung (1982h3 The SOS chromotest: direct assay of the expression of gcnc sfiA as a measure of genotoxicity of chemicals. Biochimie. 64. 797-801. Quillardet, P.. C. de Bellecombe and M. Hofnung (1985) the SOS chromotest. a calorimetric bacterial assay for genotoxins: validation study with X3 compounds, Mutation Res.. 147. 79-85. Sakai. M., Y. Daisuke and S. Mizusaki (19%) Mutagenicity of polycyclic aromatic hydrocarbons on Saimodh Qphimurium TA97. Mutation Res.. 156. 61-67. von der Hude. W., C. Behm. R. Giirtler and A. Basler (19x8) Evaluation of the SOS chromotest, Mutation Res.. 203.
x1-94. Walker, G.C. (1984) Mutagenesis and inducible response to desoxyribonucleic acids damage in Escherichia CO/~.Microbiol. Rev.. 48. 60-93.
Mtrurwr7
MLJTGEN
Olh~-I~IR/Y7/%0S.00
017X
Genotoxicity
of polycyclic aromatic V. Mersch-Sundermann,
lnsriiurr
of Medical
hydrocarbons S. Mochayedi
in Escherichia coli PQ37
and S. Kevekordes
Microbiology und Hygienr, Faculty of Clinical Mrdicine Munnhrim.
Unil yrsiry
D-6X00 Munnheim IF. R. G.) (Received
1 November lYY1))
(Revision received 2 July 1991) (Accepted 9 July 1901)
Keywords: SOS chromotest; Polycyclic aromatic hydrocarbons; Genotoxicity
Summary In the present investigation, 32 pol;cyclic aromatic hydrocarbons (PAHs) were tested for genotoxicity in E. coli PQ37 using the standard tube assay of the SOS chromotest. PAHs such as benzo[glzi]fluoranthene, benzo[j]fIuoranthene, benzo[a]pyrene, chrysene, dibenzo[a,l]pyrene, fluoranthene and triphenylens exhibited high genotoxicity when incubated in the presence of an exogenous metabolic activation mixture. ‘The results were compared to those obtained with the Salmonella/microsome test.
In E. co/i PQ37 the /3-galactosidase gene (lucZ) is placed under control of the s&l gent operon, which belongs to the SOS-repair system ($4 : : 1acZ fusion). Accordingly, upon the induction of DNA damages not only the s&f gene, but also the 1acZ gene, is expressed. Hence, the gene product of lacZ, P-galactosidasc, can be measured by a simple assay and thus provides a measure of genotoxicity. In contrast, to obtain a measure of a generalized bacteriotoxic effect, the alkaline phosphatase activity of E. co/i PO37 can be measured as it is an indicator of general protein syn?hesis (Quillardet et al., 1982a, b, 1985; Quillardet and Hofnung, 1985; Hofnung et al., 1989; Walker, 19841.
Correspondence: Dr. Volker Mrrsch-Sunderman% lnstitut fiir Medizinische Mikrobiologie und Hygiene, Fakultit fir Klinische Medizin Mannheim der Universitlt fach 1001123, D-6800 Mannheim I (F.R.G.).
Heidelberg,
Post-
As part of a continuing characterization of the E. co/i genotoxicity assay (SOS chromotest) (Mersch-Sundermann, 1989a, b), we examined the responses of 32 polycyclic aromatic hydrocarbons (PAHsJ. Materials and methods Chemicals
The 32 PAHs were tested by the standard SOS chromotest with slight modifications. The sources of chemicals are described in Table 1. The PAHs selected for genotoxicity testing are representative of those that are abundant in environmental samples (Brune et al., 1979). For testing the PAHs were dissolved in DMSO up to doses of 39-20000 rig/assay or to the limit of solubility in 10 differcnt dilutions. All chemicals were routinely tested in triplicate in the presence and absence of SO derived from the livers of Aroclor 1354-induced rats.
with
Wc follo\v~d :he procedure recommended by Quillurdct and Hofnung ( 1985) with slight modifica:ions. I ml ctf an E. I-V!! PO37 overnight culture \\;is diluted :;ith 9 ml L-riedium. Then hOl)-~.~l portions of this dilution ; = 5 X lOh cfu) we c distributed into tubes containing 20 +I of gradual dilutions of the PAHs to be tested. Using the text procedure with metabolic activation S ml of a modified S9 mis ~5OYi 52) mix) replaced 9 ml of L-me&m (see below). These mistures were incubated for 2 h at 37 OC
T.ABLE
I
CtlAR;\(‘TERIZ(\TIC)N
OF P.i\t?h
Anthanthrene Xnthracrnr Eirnz[tr]anthraccne Benzo[ h]fluoranthenr Brnzo[ g/n]iluoranthcnr Benzo[ j]tlrwrunthcnc Brnzo[~rjIluorenr Bcn,w[ h]tluorznc Benzo[ ,gIzi]pr+ne Bcn~ti[ cr]p)rrnc‘
32.32 'i7 _. _ 12 2lh.‘S 2lh.2S 77fb.34 75-l _. -..i
Benzo[eJpyrene Chnsrnr Coronrne
3’ -._3 -_ “Y ._‘Y __.
Dihrnz[u.c.];lnthrctcrn~
27s.1 27S.J
Dihrnz[tr.ll]anthracene Diiiazo[ [l.l]pyrenr Di‘nrnzo[ tr.h]pyrrnr Dihenzo[ ~.i]pqrrnr 7. I?-Dimethylhenz[u]anthracrnr
.Wl).3l
32.4 302.4
Jacob Srrw 13675 Srrva l-1530
YY’;
Aldrich .27.533-b
> 995 > YYci YWi YXri > YXri YX’i YYri Y5Ci 97% YX? YV-i
Jacob J;icoh Fluka 11-190 Fluka 124’!5 Aldrich B YOO-Y Serb3 IJxoo Aldrich B1.010-2 Aldrich CX.OOO-X Aldrich CX.GW I Serva 19275 Serva lY7XO
> YYci
Jacob
301.1
> YYci 9X?
Jacob Sigma DO33
2Sh.35
> W’i
Fluka
3.bDimethklphenanthrenr Fluoranthene Fluorrnr Indeno[ 12.3~ctllpyrenr 2-Methylanthracene Y-blcthylanthracenr
Yx’; YSCi
> YYc;
39570
Aldrich 26.190-3 Servu ‘I539 Selva 2155? Jacob
! Y23
Atlanta
I.3533
Atlanta
7121
%Mrthylcholltnthrene
I913 35X.3h
2-Mrthylphcnanthrenr Naphthalenr PU$UW
I’)‘.76 11X.1 157_.__ 3’ _.
Aldrich
Phenanthrene Pyrrnr
17x.1 ‘01.3 ‘3.‘Y
Srrva
Triphrnylrnr
Fluka
hh’30
Pl.l20-4 32005
Serva 33hh5 Aldrich
Metabolic actirution In contrast to the procedure described by Quiilardet and Hofnung (1985) we used S9 mix containing only 50% of the standard 9000 x g supernatant (see also Marzin et al., 1986). The composition of the S9 mix has been described by McrschSundermann et al. (199lb).
7b.11)4-6
Sigma N3XO Aldrich
agitatic>n. To prevent measurement problems due to the turbidity of the S9 mix a short centrifugation step (2500 x g. 5 min) followed. 550 ~1 of the supernatant was decanted carefully and the rcmainir,g bacterial pellets were resuspended in 550 ~1 of a 0.9% NaCl solution. After mixing thoroughly, a further centrifugation step with decantation of 550 ~1 supernatant and resuspension with 550 ~1 0.9% NaCl solution followed (washing step). Then. 300~~1 portions from each tube were drawn and placed in fresh tubes, such that the-.e were 2 equal series of tubes each Tontaining a volume of 300 ~1. To determine the induced P-galactosidase activity, 2.7 ml of bg buffer and 600 ~1 of 0.4% 4-nitrophenyl-fi-o-galactopyranoside !QNPG) solution were added to each tube of one series. The mixtures were incubated for 30 m;n at :‘7 o C in a water bath. The incubation was stopped with 2 ml of I M sodium carbonate. The absorbance at 405 nm was read against a blank containing all ingredients but lacking bacteria. The determination of alkaline phosphatasc activity was similar to the P-galactosidase assay except that ap buffer replaced the bg buffer and a 0.4% 4-nitrophenyl phosphate (PNPP) solution replaced the ONPG solution. The conversion of PNPP was stopped with 2 ml of I.5 N sodium hydroxide. Positive controls, equipments and sources have been described elsewhere (Mersch-Sundermann et al., 1989a, b, 19Yla). The photometer was a Hitachi model 1170-60 digital double-beam spectrophotometer (horizontal beam). Measurements were made with 1 cm standard plastic cuvettes.
TKXO-0
Calculation The P-galactosidase (bg) and al!:sline phosphatase cap) activities were calculated according to the simplified version recommended by Quillardet and Hofnung (1985): units = A405 x
TABLE 7 GENOTOXlCiTY Compound
OF P.Ws
IN Eschrrichiu
Dose
(/.&assay)
coli PO37
(TUBE ASSAY) IN = 3)
Assay ( - S9J
Assay ( + 94)
Pgai
ap
(units)
(units)
0.156 0.625 2.500 10.000
5.25 5.63 5.73 6.18 6.19
Anthracene
0.000 0.156 0.625 2.500 10.000
Benzlalanthracene
IF
SOSIP
17.4; 17.37 17.41 17.29 i7.:5
1.oo 1.08
0
5.19 5.23 5.23 5.23 5.25
0.000 0.156 0.625 2.500 10.000
Penzo[ h]fluoranthene
&gal iunits)
av (&iitsJ
ratio
SOSiP 0.132
I .09 1.19 1.20
9.63 9.51, 8.97 4.84 8.73
1.uu
5.49 h.24 10.37 13.80
1.25 2.13 2.89
16.88 lb.84 16.81 16.76 16.20
1.00 1.01 1.01 1.02 1.05
5.37 5.56 5.91 6.31 6.41
9.33 9.09 8.92 x.77 8.19
1.00 1.06 1.15 1.25 1.36
0.010
4.57 5.01 4.99 5.03 5.31
lb.27 16.13 16.12
5.32 5.49 5.79 6.96 18.91
8.85 8.73 8.73 8.67 8.65
1.00 1.05 1.11 1.34 3.64
O.!%J
16.16
1.00 1.11 1.10 1.10 1.18
0.000 0.156 O.b25 2.500 10.000
5.45 5.48 5.52 5.49 5.15
17.25 17.15 17.19 17.19 16.89
1.oo 1.01 I .02 1.01 0.96
5.49 5.95 6.41 8.12 14.76
9.03 8.95 8.91 X.81 8.69
1.OtJ 1.09 1.18 1.52 2.79
0.045
Benzo[ghi]fiuoranthene
o.oon 0.155 0.625 2.500 10.000
4 Y3 5.08 4.99 5.05 5.09
16.15 16.08 16.16 16.09 16.01
1.oo 1.03 1.01 1.03 1.04
5.40 5.63 6.55 12.51 33.40
9.56 9.41 9.08 X.97 8.84
1SHJ I .06 1.28 2.47 6.h’)
0.340
Benzo[ j]fluoranthene
0.000 0.156 0.625 2.500 10.000
4.72 5.39 5.45 5.41 5.40
16.64 16.56 iii.45 16.56 16.68
1.oo 1.15 1.17 1.16 1.15
5.07 6.00 6.85 15.77 19.15
9.21 8.89 8.76 8.67 8.32
1.00 1.23 1.42 3.31 4.18
0.254
Benzo[ rc]fluorene
0.000 0.156 0.625 2.500 io.c00
4.96 5.40 5.37 5.43 5.48
lb.97 16.49 16.52 16.48 16.49
1.00 1.12 1.11 1.13 1.14
4.97 4.91 4.88 5.00 5.00
X.56 8.56 8.52 8.57 8.59
i .uo 0.99 0.94 1.oo 1.0
0
Benzo[ hlfluorene
0.000 0.156 0.625 2.500 10.000
5.32 5.41 5.41 5.3’) 5.45
17.37 17.35 17.33 17.32 17.29
1SJO 1.02 1sJ2 1.02 1.03
5.04 5.45 5.91 6.80 9.61
8.43 X.28 8.11 7.07 7.ni
1.oo I.10 1.22 1.43 2.tJ6
0.024
Benzo[ ghi]perylene
0.000 0.312 1.250 5.000 10.000
5.33 5.39 5.37 5.43 5.39
17.19 17.15 17.08 17.05 17.13
1.oo 1.01 1.02 1.03 1.01
4.85 5.83 6.05 7.25 9.15
8.85 8.28 8.03 7.84 7.X0
i .OO 1.29 1.38 1.69 2.14
0.033
Anthanthrene
0.000
16.03
5.35
1.04
Campound tnclmc)
Benzo[a]pyrenr
Ben& r]pyrene
Chrysene
DWX (j.lg/assay)
Dibenz[a.c]anthracene
Dibenz[o,/r]anthracene
B-gal (unitsi
0.243
5.02 6.22
9.19 8.99
1.00 1.27 2.17
16.72 15.83
1.oo 1.15
0.625 2.500
5.91 6.17
15.87 15.87
1.30 I .36
10.25 24.91
8.67 8.54
10.000
6.28
15.63
1.41
31.13
8.34
0.000 0.156
5.13 5.17
17.15 17.05
I .oo 1.01
4.79
8.60
0.625 2.500 10.000
5.21 5.25
17.08 17.07
1.02
5.43 5.53
8.40 8.27
5.21
17.07
1.03 1.02
6.63 6.93
8.16 7.91
0.000 0.156 0.625
4.16 4.hO
15.25
1.oo
15.05 15.05
1.12 1.14
5.12 5.88
8.55 8.39
15.04 14.92
1.16 1.17
6.32 12.79
8.27 8.09
35.59
8.00
15.81
1.00 106
5.21
8.97
5.65 5.75
8.85 8.88 8.72
4.68 4.75 4.77
0.000 0.05 I
4.39 4.48
0.206 3.825 3.300
4.49 4.52 4.44
15.31 15.35 15.40 15.36
0.000 0.156 0.625
4.72 4.85 4.88
16.09 16.12
2.500 10.000
4.85 5.56
16.04 15.93
0.000 0.156
4.68 4.84 5.09 5.12 5.15
0.000
4.55
pyrene
0.156 0.625
5.05 5.11
2.500 10.000
4.85 5.01
0.000 0.052 0.206 0.825
5.47 5.47 5.17
3.300 Dibenzo[Q,i]pyrene
P-gal (units)
5.21
Dibenzo[a,i]-
pyrene
SOSlP
4.79
0.625 2.500 10.000
Dibenzo[Q,h]-
ratio
IF
O.OOil 0.156
2.500 10.000 Coronrne
Assay ( + S9)
Assay ( - S9)
0.000 0.156 0.625 2.500 10.000
16.39
1.05 1.06 1.04
6.13 6.85
8.41
1.00 1.16
1.58 1.00 1.17 1.28
1.00 1.10
1.00 1.10
1.05 1.05
6.15
8.73 8.37
1.22 1.74
8.12
2.99 1.oo
5.24 5.55
9.29 9.08
16.20 16.03 15.93
1.09 1.11
5.60 6.76
8.92 8.51
1.13
11.05
8.29
16.43 16.13
1.00
5.12 10.08
1.41
1.oo 2.02
7.85 7.79
12.55
1.00
16.11
1.09
20.49 55.67
16.09
1.13
46.35
5.39
17.33 17.23 17.28
1.oo 1.01
5.38
5.40
5.79
9.80 9.59
1.02
10.54
6.21 6.93
9.37 8.95
1.oo
10.35
8.79
5.04
17.25
8.87
1.00
16.84
1.oo 1.03
5.09
5.07 5.13
8.79 8.77
1.13
1.04
5.68 7.56
1.05 1.09
12.31 18.99
8.75
1.50 2.45
8.35
3.96
5.35
16.76
0.117
1.10 1.21
1.02
16.88 16.81
2.100
4.15
17.27 16.56
5.17
0.039
2.36
9.05
16.13
0.104
1.08 1.11
8.84 n.75
1.13 1.14
0.027
1.11 1.21 1.40
8.97
1.oo 1.04
0.221
2.64 7.43
9.24
16.32 16.25
0.032
1.20 1.46
5.33 5.69
1.21
0.605
5.34 6.84
1.oo 1.05
8.41 14.01
SOSIP
1.41 2.14 0.174
TABLE 2 (continued) Compound (name)
Dose (/Lg/assay)
Assay t - SY)
Assay ( + S9)
P-gal (units)
aP (units)
IF
5.07 5.65 5.71 5.73 6.01
17.17 16.56 15.52 15.33 15.33
1.00 1.16 I .25 I .27 1.33
0
5.29 5.69 6.44 X.51 i5.80
9.07 8.91 X.80 x.53 x.44
1.00 I 0’) 1.25 1.71 3.21
0.072
0.156 0.625 2.500 10.000
3,6-Dimethylphenanthrene
0.000 0.156 0.625 2.500 10.000
4.89 5.16 5.19 4.60 3.84
16.88 16.76 16.83 16.24 15.55
1.00 I .07 1.07 0.98 0.86
0
5.43 5.72 6.47 6.77 7.71
9.55 9.4x 9.47 9.47 9.47
1.00 1.06 1.20 1.26 1.43
0.007
Fluoranthene
0.000 0.156 0.625 2.500 10.000
5.01 5.04 5.07 5.11 5.09
16.84 16.71 15.52 14.11 14.04
1.00 1.02 1.19 1.22 1.22
0
5.32 7.16 10.95 30.32 40.57
9.11 X.91 x.75 8.57 8.24
1.oo
0.4 I3
0.000 0.156 0.625 2.500 10.000
4.79 4.89 4.85 4.63 3.47
17.05 16.08 16.09 14.47 9.25
1.00 1.08 I .08 1.14 1.33
0
5.16 5.16 5.16 5.12 5.11
8.79 8.81 8.60 8.89 8.73
0.98 1.00
Indeno[ 1,2,3-cd]pyrene
0.000 0.156 0.625 2.500 10.000
5.59 5.65 5.75 5.39 5.21
17.31 17.29 17.27 17.09 17.03
1.oo I .02 1.03 0.98 0.95
0
5.15 5.31 5.83 6.75 10.35
9.0 1 8.96 8.93 8.84 x.97
1.00 1.04 1.14 I .34 2.02
0.036
2-Methylanthracene
0.000 0.156 0.625 2.500 10.000
5.11 5.46 5.52 5.52 5.49
17.49 17.37 17.47 17.40 17.41
1.oo 1.08 1.08 1.09 1.08
0
5.11 5.77 6.53 9.01 9.81
9.13 9.04 E.85 8.75 8.33
1.OO 1.14 1.32 1.84 2.11
0.052
9-Methylanthracene
0.000 0.156 0.625 2.500 10.000
5.16 6.20 5.45 2.28 1.23
17.40 17.31 16.08 5.52 2.67
1.oo 1.20 1.14 1.39 1.55
0
5.16 5.60 6.32 10.88 13.87
9.53 9.29 9.16 8.93 8.78
I .oa 1.11 1.27 2.26 2.93
0.102
3-Methylcholanthrene
0.000 0.156 0.625 2.500 10.000
4.80 5.63 5.64 7.44 11.25
17.05 16.32 16.29 16.35 16.03
1.00 1.23 1.23 I .62 2.50
0.036
5.41 5.80 9.25 18.96 22.03
8.92 8.88 x.77 8.69 x.53
I .oo I .08 1.74 3.59 4.25
0.194
2-Methylphenanthrene
0.000 0.156 0.625 2.500 10.000
4.95 6.00 5.83 1.57 1.37
16.47 16.37 15.36 2.92 2.65
1.oo 1.22 1.26 1.79 1.73
0
5.43 6.12 7.28 10.92 9.08
9.39 9.21 9.35 9.25 9.31
1.00 1.15 I .35 2.04 I .6Y
o.ah8
7,12-Dimethylbenz[a]anthracene
Fluorene
0.000
SOSIP
P-gal (units)
aP (units)
ratio
SOSIP
1.38 2.14 b.06 8.43 1.oo
0
I .oo 1.02
Naphthalcne
-l.c)b
:7.1
I .oo
S.-u 5.20
16.7’) I(,.09
I.12 I.12
5.4x 5.4X
5.15 4.h4
I-l35 I I .85
I.20 I.36
5.11
5.20
17.37 17.29 17.11 17.OY
I .oo
s.3 5.29 5.27 S.23 Phrnanthrrnr
0
1)
I .02 I si3
5.01 5.h’)
1.32
lh.33 lb.15
I .oo 1.04 I.10 I.11
-1.89
15.0x IS.09 15.04
5.35 5.48 5.60
I.10
S.Yh
S.2-I _5 ._77
17.21 17.16
I .oo I.01
5.3 I
I7.04 17.13
I .02
h.0’) 7.4x
I.01 I .07
17.79 7’ 9’) __.
Ih.24
0
I .03
1000/r (A405 = optical density at 405 nm; t = substrate conversion time in minutes). The induction factor IF was calculated as the ratio Rx/R,, (R, = bg/ap determined for the test chemical; R,, = bg/ap determined for the negative control). The SOS-inducing potential (SOSIP) that describes a single and comparable parameter for the genotoxic potency of a compound was determined as the steepest slope of the IF dose-response curve (increase of IF per nmole of compound). A compound was classified as ‘non-reactive’ (‘not genotoxic’) if the induction factor remained < 1.5, as ‘marginal’ if the induction factor was between 1.5 and 2.0 and as ‘reactive’ (‘genotoxic’) if the induction factor was in excess of 2.0. Additionally, to avoid false-positive results due to bacteriotoxic effects (decrease of ap activity), a further requirement for genotoxicity was a
0
0.0 I 7
6.71
4.35
5.24 5.29
SOSIP
h.05 6.49
I .03 I .t17
I .03 1.34
-I.% 1.x’) 4.c)Y
ratio
5.43
IS.95 IO.39
4.81
ap (units)
S.48
I .oo
4.28 1.7h
Triphenylene
1h.h’)
(units)
lb.57 1b.M
511 S.23 5.07
Pyrene
I
SOSIP
,fz-gal
3P (units)
IF
(units)
p-g.31
S.!)7 5.31 6.49
0.053
K-3 I%4 0
0
continuous compound
>.73
5.16
0
0.260
increase in bg activity with increasing concentration.
Results and discussion The genotoxic activities of 32 PAHs on E. coli PQ37 are shown in Table 2. Anthracene (SOSIP = 0.01) (Fig. lA), benzo[a]fluorene (SOSIP = 0.0241, coronene (SOSIP = 0.027), 3,6-dimethylphenanthrene (SOSIP = 0.0071, fluorene (SOSIP = 01, naphthalene (SOSIP = O), perylene (SOSIP = 0.017) and pyrene (SOSIP = 0) showed little or no genotoxic activity (IF < 1.5) either in the presence or in the absence of S9. In addition benzo[e]pyrene (IF (max) = 1.9, SOSIP = 0.032) displayed a weak response. All the other compounds induced the SOS system of E. co/i PQ37 in the presence of S9 mix.
7
The highest activity was shown by dibenzo[~~./]pyrcne with a SOSIP = 2.1 (IF tmax) = 12.5) (Fig. 10. In the absence of a metabolizing system only benzo[a]pyrene (SOSIP = 0.243) and 3-methylcholanthrene (SOSIP = 0.036) showed genotoxic effects. The influence of the USCof S9 mix containing less 9000 x g supernatant from Aroclor 1254-induced rat livers than the standard S9 mix on the SOS response of E. co/i PQ37 is shown in Fig. 2. Whereas we had only a very weak or questionable
response testing O-634 ng benzo[alpyrcne/assay using the standard SC)mix (54 ~1 9000 x g supernatant/assay), the response was increased (up to IF = 2.0) (reactive) when using the ‘50% SC)mix (27 ~1 Sc)/assay) and only 312 ng benzo[a]pyrene/assay. Additionally the SOSIP was significantly higher using lower SC)concentrations (data not shown) (Mersch-Sundermann et al., 199lb). The cause is probably the inactivation of trace amounts of /I-galactosidase by components present in the 9000 X g supernatant. The SOS re-
induction factor 3.0
units A
units
Anthracene (+ S9)
15
inductton factor - 3.0
B Anthanthrene
15
(+ S9)
t
I; si
2.5
10 -.
10
0
2.0
. . 2.0
5 .. . . 1.5
1.5
0 .-
1.0 i
I
500
SOSIP = 0.142
. . 1.0
I
:,:~::~:.::~:~::~::‘:::‘!:
0
. . 2.5
t
1000 1500 nglassay
2000
_.0
2500
. 500
1000 1500 nglassay
2000
2500
induction facto! Dlbenzo(a,l)pyrene
I
+ + +
Fig. I. P-Galactwidase (nonreactive
compound),
and ;dkaline
:,
500
:: ::::,z: :::,:: 1000 1500 2000 nglassay
’ 2500
R-galactosidase activity alkaline phosphatase activity induction factor
phosphatnse
(5) anthanthrrnr
‘:;:’
0
(+ S9)
activity.
(reactive
calculated
compound)
induction
factor
and SOS inducing
and (C’) dihenzo[a.l]pyrcne
(extremely
potential
of (A) anthracent:
highly reactive
compound).
x inditctton 5--
factor
but only verified for the testing of PAHs (Mersch-Sundermann et al., 199lb). A comparison of the SOS chromotest results with those obtained in the Salmonella/microsome assay (Sakai et al., 1985) for 18 PAHs showed a good correspondence between genotox-
4-
3-
2r
TABLE
3
GENOTOXICITY
AND
MUTAGENICITY
OF PAHs
i-
Name
i
Q10
20
30
40
90009
supernatant
B(a)P
50
60
70
80
concentration
0.078
llg
4
0.156
ug
-i-
0.625
Ug
-
1.25 “9
-
0,312
-
2.5
tlg
ug
Fip. 2. Influence of the 9000 X !: supernatant concentration on the response of E. colr PQ37 testing hrnzo[a]pyrene.
sponse is further increased when using very low SC) concentrations (6 PI/assay) and a low compound concentration (Fig. 2). It is to be noted that the increase of SOS-inducing activities when using this modified S9 mix is not a general rule,
induction factor
8 testing5000
nella assay a
nglaasay 0 fluoranelerle
Anthanthrene
2.89
Anthracene Benz[a]anthracene
1.36 3.78
Benzo[b]fluoranthene Benzo[ g&]fluoranthene
3.21 8.11
BenzoIjJfluoranthene Benzo[ alfluorene Benzo[ blfluorene
4.44 1.01 2.23 2.14
Benzo[ &:lli]perylene Benzo[ cllpyrene Benzo[ rjpyrene
6.94 I .YO
Chrysene Coronene
7.43 I .47
Dibenz[u.c]anthracene
2.99
Dibenz[a,h]anthracene
2.62 12.55
Dibenzo[ a,l]pyrene Dibenzo[cr,h]pyrene
2.62 4.26
anthracene 3.6-Dimethylphenanthrene
3.66
Fluoranthene Fluorene
x.71
I so I .02
6
Indeno[l,2,3-cdlpyrene
2.59 2.35
5
2-Methylanthracene Y-Methylanthracene
8 Chrysene
2.93 4.84
3-Methylcholanthrene
2.04
2-Methylphenanthrene Naphthalene 7.1%Dimeth benz(a)amhacene e QMet 1cylanthracene Phenanthrene 0 2-Methylanthracene 2-MethylPhenanthrene ti @ Benzo(ghi)perylene
3 2
1.OY
1.46
Perylene Phenanthrene Pyrene Triphenylrne
+ _ + + + + + + + (+) + _
+ - 1) + + + + ? ‘> + + + + + +
+ + + + +
0 + +
+ -
+ _
+ _
+ -
+ + + + + -
+
2.94 I.21
-I-
5.03
+
-
+
+ + + 0 _ + - 1) + +
’ Data I’rom McCann et al. (1975), Brune et al. (1979), Bartsch et al. (1980). Haworth et al. (1983). Sakai et al. (1985).
0 0
100
200
cr::::::::::::::::::::::: 300 400
Brahms et al. (1987).
500
( + ) weak genotoxicity (I.5 < IF > 2.0); 0 data inadequate or
SJyphimurium TA97 revertants per plate Fig.
genotoxicitv
Dibenzo[u.i]pyrene 7.12-Dimethylbenz[a]-
7
Salmo-
IF (max) 90
(@assay)
-
SOS chromotest
3.
Correlation
of genotoxicity
on E
not available; ? conflicting reports; I) negative in TAl535, coli
PQ37
mutagenicity on Sulmonrlla ryphimurium TAY7.
and
TA1537, TA1538, et al.. 1385).
TA98,
TAlOO;
positive only TA97 (Sakai
icity in E. coli PQ37 and mutagenicity in Salmonella typhimurium TA97 (Fig. 3) (Table 3). In view of this and other studies (von der Hude et al., 1988) the SOS chromotest seems to possess some practical advantages over the widely used Salmonella,/microsome assay (Maron and Ames, 1983). The SOS chromotest is easy to perform, the results are obtained within a few hours and only a single bacterial strain is required. Thus, further characterization of the assay with respect to the response of diverse chemical classes, the standardization of the test protocol and the determination of the structural basis of SOS-inducing activity are warranted to evaluate the role of the SOS chromotest as part of a battery for the prediction of carcinogenicity.
Press, London. pp.
Maron. D.M.,
and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test. Mutation Rcs.. 113.175.-21.5. Marzin.D.R., P. Olivier and H. Vophi (1986) Kinetic detcrmination of enzymatic activity and modification of the metabolic activation system in the SOS chromotest. Mutation Res., 164, 353-359. McCann. J.. E. Choi. E. Yamasaki and B.N. Ames (1975) Detection of carcinogens as mutagens in the Salmonella/ microsome test: assays of 300 chemicals. Proc. Natl. Acad. Sci. (U.S.A.), 72, 5135-5139. Mersch-Sundermann.
V., A. Hofmeister.
G. Miiller
and H.
Hof (l989a) Untersuchungen zur Mutagenitat organischer Mikrokontaminationen in der Umwelt. III. Mitteilung: Die Mutagenitat ausgewahlter Herbizide und lnsektiride im H.
Hofmeister
(1989b) Untersuchungen zur Mutagenitat organischer Mikrokontaminationen in der Umwelt. IV. Mitteilung: Die
We thank Herbert S. Rosenkranz from the Department of Environmental and Occupational Health at Pittsburgh, PA for his advice and the reading of our manuscript. Moreover, we thank P. Quillardet and M. Hofnung from the Institut Pasteur, Paris, for the Escherichia coli strain PQ37 and J. Jacob from the Biochemical Department for Environmental Carcinogens, Ahrensburg, for a number of the test compounds. Furthermore, we thank W. Meister for his laboratory help.
Mutagenitiit leichtfliichtiger Organohalogene im SOSChromotest, Zbl. Hyg., 189. 266-271. Mersch-Sundermann. V.. S. Kevekordes and S. Mochayedi (1991a) Sources of variability of the Esrhericl~iu coli PQ37 genotoxicity assay (SOS chromotest). Mutation Res.. 252. 51-60. Mersch-Sundermann. V.. F. Wintermann and S. Kern (199lb) Sources of variability of the SOS chromotest: influence of the metabolizing system on the response oi E. ct,li PQ.77. in press. Quillardet. P.. and M. Hotnung (19X5) the SOS-Chromotest. a calorimetric bacterial assay for genotoxins: procedures, Mutation Res., 147, 65-78. Quillardet. P.. 0. Huisman. R. d’Ari and M. Hofnung (1982a) SOS chromotest. a direct assay of induction of an SOS
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Brams, A., J.P. Buchet. M.C. Crutzen-Fayt,
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C. de Meester, R.
Lauwerys and A. Leonard (1987) A comparative study. with 40 chemicals, of the efficiency of the Salmonella assay and the SOS chromotest (kit procedure). Toxicol. Lett., 38, 123-133. Brune, H., R. Deutsch-Wentzel,
S. Dohbertin.
G. Grimmer,
M. Habs. J. Jacob J. Misfeld, U. Mohr. G. Obersdoerster. F. Pott, D. Schmaehl, P. Schneider and D. Steinhof (1979) Luftqualitatskriterien fiir ausgewlhlte polyzyklische aromatische Kohlenwasserstoffe, Erich Schmidt, Berlin. S.. L. Lawler.
Genetic Risk Assessment, Academic 325-136.
SOS-Chromutest. Zbl. Hyg., 189, 135146. Mersch-Sundermann, V., G. Miiller and
Acknowledgements
Haworth,
Hofnung. M.. P. Quillardet. 0. Goerhch and E. Touati (19x9) SOS chromotest and the use of hack% for the &tectjon and diagnosis of genotoxic agents. in: bie7.v Trends ;n
K. Mortelsman,
W. Speck and E.
Zeiger (1983) Salmonella mutagenicity test results for 250 chemicals, Environ. Mutagen.. Suppl. I. 3-142.
function in Eschericlri~ co/i K-12 to measure gecotoxicity. Proc. Natl. Acad. Sci. (U.S.A.!, 79, 5971-5975. Quillardet, P., 0. Huisman. R. d’Ari and M. Hofnung (1982h3 The SOS chromotest: direct assay of the expression of gcnc sfiA as a measure of genotoxicity of chemicals. Biochimie. 64. 797-801. Quillardet, P.. C. de Bellecombe and M. Hofnung (1985) the SOS chromotest. a calorimetric bacterial assay for genotoxins: validation study with X3 compounds, Mutation Res.. 147. 79-85. Sakai. M., Y. Daisuke and S. Mizusaki (19%) Mutagenicity of polycyclic aromatic hydrocarbons on Saimodh Qphimurium TA97. Mutation Res.. 156. 61-67. von der Hude. W., C. Behm. R. Giirtler and A. Basler (19x8) Evaluation of the SOS chromotest, Mutation Res.. 203.
x1-94. Walker, G.C. (1984) Mutagenesis and inducible response to desoxyribonucleic acids damage in Escherichia CO/~.Microbiol. Rev.. 48. 60-93.