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Genotoxicity of dental materials
Genetic Toxicology
ELSEVIER
Mutation Research 368 (1996) 181- 194
Genotoxicity of dental materials JiJrgen Heil a,*, Georg Reifferscheid a, Petra Waldmann a, Gabriele Leyhausen Werner Geurtsen b
b,
a AMMUG, Universit~it Mainz, Obere Zahlbacher Str. 63, D-55101 Mainz, Germany h Poliklinikfiir Zahnerhaltung und Parodontologie. Medizinische Hochschule Hannol,er, Konstanty-Gutschow-Str. 8, D-30625 Hannoz,er, Germany
Received 8 January 1996; revised 1 February 1996;accepted 2 February 1996
Abstract This study was performed to characterize the (possible) DNA-damaging properties of dental materials and to identify specific compounds that contribute to this genotoxicity. For screening, three tests that assay for different aspects of genotoxicity (i) the bacterial umu-test; (ii) the eucaryotic DNA synthesis inhibition test; and (iii) the in vivo alkaline filter elution technique were chosen. This investigation gives several lines of evidence that most dental materials tested (14 chemical monosubstances present in dental devices and 7 extracts of dental materials) yield 'positive' results in at least one of the genotoxicity tests, however, with effects ranging from 'borderline' to 'strong positive'. The extracts of the widely used dental materials Vitrebond® and AH26 ® elicited clear concentration-related genotoxic responses in all test systems. On the basis of these data and public concern, more attention has to be given to local or systemic complications which may be associated with the use of dental materials. Kevwords: Genotoxicity;Dental material; Umu-test; DNA synthesis inhibition;Alkaline elution technique
1. Introduction Since 1995 dental materials are classified as medical products. Therefore, it is indispensable according to national as well as international regulations that medical devices - either newly developed or currently in use - have been proven for biocompatibility and that their toxicological profile has been determined. Previous studies have shown that dental materials may induce local and systemic adverse effects which are caused by extractable monomers
* Corresponding author. Tel.: +49-6131-173314; Fax: +496131-173364
(Spahl and Budzikiewicz, 1994; Spahl et al., 1991) a n d / o r other inorganic and organic ingredients. Various in vivo and in vitro test systems have been used in order to identify the components responsible for the reduction of the biocompatibility of these materials (Lehmann et al., 1993). Meanwhile, there are some few papers dealing with the genotoxic activity of selected dental materials (Leyhausen et al., 1995; Schweikl et al., 1994; Stea et al., 1994), mostly Ames test data. Out of 12 commercially available dental cements, Stea et al. found one cement clearly mutagenic to Salmonella typhimurium; however, with nearly all cements showing strong bactericidal activity. This fact, among
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182
J. Heil et al. /Mutation Research 368 (1996) 181 194
others, indicates that bacterial test systems cannot be the only basis for the assessment of the DNA-damaging properties of medical products. This study, which came into being by a close cooperation of genetic toxicologists with dentists, reports the outcome of genotoxicity studies of some widely used dental materials and their components. To our knowledge, this is the first compilation of results obtained with three test systems covering different steps of mutagenesis/ carcinogenesis: (i) the bacterial umu-test, monitoring the mutagenic potential of a substance; (ii) the eucaryotic DNA synthesis inhibition test, detecting potential carcinogenic substances; and (iii) an in vivo alkaline filter elution technique, sensitive to strand breaks. These special methods - primarily developed by our group for the testing of environmental samples and in successful use for several years - have been combined to a battery of genotoxicity tests which is fast, easy to perform and statistically reliable.
tration in medium was set to 1.2% (cell culture test) resp. 3% (bacterial test).
2. Materials and methods
2.2.2. Extracts of dental materials Dental materials tested are listed in Table lB. These materials often consist of about 10-20 single ingredients, mainly anorganics (e.g., SiO 2, ZnO, Na3AIF6, A1203, Bi203, Ca(OH) 2, Ca3(PO4) 2, TiO 2, YF3), various methacrylates and others (e.g., initiators, inhibitors, color pigments, natural oils). All materials were prepared according to the manufacturer's instructions. Briefly, 300 mg of each root canal filling material (AH26 ®, N2 ®, Apexit ®) were mixed and placed in screwcaps of polyethylene vials and set for 3 days at 37°C and 100% humidity. Composites (Herculite XR ®, Sono-CEM ~) and glass ionomer cements (Ionoseal ®, Vitrebond ®) were mixed and polymerized in silicone molds for 60 s by a blue light source (Elipar II, Espe, Seefeld, Germany). Thereafter, the hardened specimens were extracted with 5 ml of serum- and antibiotics-free cell culture medium or 5 ml of DMSO for 24 h at 37°C. Extracts were stored frozen at - 2 0 ° C until use.
2.1. Materials
2.3. Bacterial test system
Dental materials tested were aliquots from materials used for clinical purposes. Supplies (microplates, cell culture flasks) were from Greiner, Frickenhausen, Germany; cell culture media and sera were obtained from Biochrom (Berlin, Germany). Fine chemicals as well as peroxidase-conjugated antimouse IgG antibody were purchased from Sigma (Deisenhofen, Germany). Monoclonal anti-BrdU antibody was from Bio Cell Consulting (Reinach, Switzerland).
2.3.1. Umu-test The umu-test has been developed for the detection of genotoxins by Oda et al. (1985), who generously provided us with the tester strain. The test is based on the capability of genotoxic agents to induce the umu operon in Salmonella typhimurium strain TA1535/pSKI002. Since the umuC gene in the bacteria is fused with the lacZ gene for/3-galactosidase, the induction rate of umuC can be easily assessed by determination of /3-galactosidase activity. The products of the SOS-regulated umuCD operon are directly involved in both point and frameshift mutagenesis, bacteria lacking these (or functionally analogous) mutator genes can be viewed as nearly nonmutable (Battista et al., 1990). Originating from the (quite insensitive) Ames tester strain TA1535, this (sensitive) umu strain was constructed by introducing the plasmid pSK1002 into the bacteria. Like in the (sensitive) Ames tester strains TA98 and TAI00, which carry an analogous plasmid (pKM101) coding for error-prone DNA repair (SOS mutagene-
2.2. Sample preparation 2.2.1. Monosubstances The chemicals (purity > 98%) are listed in Table 1A. Most substances require the use of an organic solvent to enhance solubilization. Each substance was dissolved in DMSO at a stock concentration of 2 M. This stock solution was serially diluted in 1:2 steps and transferred onto the microplate with the tester organisms using a laboratory workstation (Merlin, Bonn, Germany). The final DMSO concert-
J. Heil et al. /Mutation Research 368 (1996) 181-194
sis), this greatly enhances survival after exposure to genotoxins; however, at the cost of an increased mutation rate (i.e., higher sensitivity of such tester strains). Not least on this account, a recent study confirmed that the results of the umu test are statistically equivalent to the Ames test (McDaniels et ai., 1990). The umu test was performed in microplates as described by Reifferscheid et al. (1991). Salmonella from stock were grown in nutrient broth for the overnight culture. Logarithmically growing tester bacteria were exposed to varying concentrations of the test material. All concentrations were tested in triplicate; with each set of experiments usually repeated three times. After 2 h of exposure, the bacterial suspension was diluted 10-fold, followed by a subsequent additional incubation period of 2 h. Thereafter, bacterial growth was measured as turbid-
183
ity (E600) with a microplate reader. The D N A damage induced expression of u m u C was quantified via the determination of /3-galactosidase activity at 420 nm using O N P G (o-nitrophenyl-/3-mgalactopyranoside; Sigma) as a substrate. In all experiments, the standard genotoxin 4-NQO (4-nitroquinoline Noxide) was used as positive control. 2.3.2. Rating o f umu test results
The results of the tests are given as mean of triplicate determinations +__standard deviation. According to the DIN (Deutsche Industrie Norm) and ISO (International Organization for Standardization) working draft ' u m u test', an induction rate of the umu gene exceeding 1.5-fold control values is scored as 'sufficient positive'. Since it cannot be excluded that concomitant cytotoxicity (growth factor < 0.5) interferes with genotoxicity assessment, these sam-
Table 1 A. List of monosubstances tested Abbreviation
Chemical composition
Function
BEMA BHT Bis-EMA Bis-GMA CQ CSA DCHA DEGDMA DIPA DMAPE DMBZ HEMA TPSb UDMA
benzylmethacrylate butylated Hydroxytoluene ethoxylated bisphenol-(A)-glycidylacrylate bisphenol-(A)-glycidylacrylate campherquinone campheracidanhydride dicyclohexylamine diethyleneglycoldimethacrylate 2,6-dilsopropylaniline 2-(4-dimethylaminophenyl)ethanol 2,2-dimethoxybenzoin 2-hydroxyethylmethacrylate triphenylstibane urethanedimethacrylate
monomer inhibitor monomer monomer photoinitiator derivative of CQ coinitiator monomer coinitiator coinitiator photoinitiator monomer derivative of Bis-GMA monomer
B. List of dental materials tested Abbreviation
Field of application (manufacturer)
AH26°~ APEXCIT ~ N2 ~ HERCULITE* SONO-CEM® IONOSEAL~ VITREBOND~
root canal filling material (De Trey, Germany) root canal filling material (Vivadent, Liechtenstein) root canal filling material (Indrag AGSA, Switzerland) light curing hybrid composite for restorations (Kerr, USA) light curing hybrid composite for restorations (ESPE, Germany) light curing glass ionomer cement for cavity lining (VOCO, Germany) light curing glass ionomer cement for cavity lining (3M, Medica, Germany)
184
J. Heil et al. / Mutation Research 368 (1996) 181-194
Table 2 UMU test and DIT genotoxicity results of chemical monosubstances identified in dental materials (for details, see Section 2: Materials and Methods) Substance
Conc.[mM]
umu-test Induction rate
Controls Negative (solvent) Positive (NQO) BEMA
0.0003
DIT Growth factor
1.0 -t- 0.03 3.0 ± 0.10
Test result
% DNA synthesis
Cell count
1.0 1.0
100 ± 8 50 ± 6
100 90
112_+2 99 _+ 7 125 _+ l0 115_+4 98 _+ 7 103 + 2
94 94 94 97 100 94
Test result
5 2.5 1.3 0.6 0.3
1.4 1.3 1.2 1.1
± 0.02 ± 0.03 ± 0.03 +0.04
< 0.1 <0.1 0.5 0.6 0.6 0.8
BHT
6 3 1.5 0.8 0.4
0.8 0.7 0.7 0.8 0.8
± 0.01 ± 0.07 -+ 0.02 -+ 0.03 ± 0.04
1.0 1.1 h1 1.0 1.0
26 ± 5 14±2 20 _+ 6 62 ± 15 97 ± 7
30 23 32 72 96
(+)
Bis-EMA
6 3 1.5 0.8 0.4 0.2
0.5 0.6 0.7 0.6 0.6 0.7
-+ 0.04 ± 0.08 ± 0.05 ± 0.08 ± 0.03 -- 0.07
2.1 2.0 1.6 1.6 1.6 1.5
39 40 60 58 70 86
59 49 49 63 60 52
+
Bis-GMA
150 75 38 19 10 5 2.5 1.3 0.6 0.3 0.15 0.08 0.04 0.02
0.9 0.5 0.5 0.5 0.6 0.6 0.8 0.8 -
± 0.02 ± 0.04 ± 0.02 ± 0.04 ± 0.02 ± 0.05 -+ 0.08 _+ 0.06
1.2 2.0 1.7 1.9 1.8 1.5 1.2 1.2
20 l0 5 2.5 1.3 0.6 0.3 0.2
4.1 3.4 1.7 1.2 1.1 1.0 1.0 1.0
± 0.21 ± 0.06 ± 0.05 _+ 0.02 ± 0.03 ± 0.01 ± 0.02 ± 0.01
CQ
10
-
+
-
0.1 0.3 0.5 0.6 0.8 0.9 0.9 0.9
± 5 ± 1 _+ 4 ± 9 ± 6 ± 5
23 ± 1 19+ 1 18+ 1 50 ± 4 123 _+ 11 105 ± 6
28 46 48 50 53 77
31 + 2 86 ± 2 91 + 5 117___2 116+7 113_+6 105 _+ 9
44 70 91 87 98 96 98
+
+
J. Heil et al. / Mutation Research 368 (1996) 181-194
185
Table 2 (continued) Substance
CSA
Conc.[mM]
20 10 5 2.5 1.3 0.6 0.3 0.2
umu-test
DIT
Induction rate
Growth factor
Test result
-
<0.1
(+)
-
<0.1
3.0±0.07 1.6±0.04 1.3±0.00 1.1±0.03 1.0±0.01 1.0±0.03
0.1 0.4 0.7 0.8 0.8 0.8
0.1
-
-
0.05
-
0.03
-
0.01
-
-
-
DCHA
20 10 5 2.5 1.3 0.6
1.3 1.1 1.0 1.0 1.0 1.0
_+ 0.01 ± 0.04 ± 0.02 _+ 0.02 _+ 0.00 _+ 0.00
0.8 0.8 0.8 0.9 1.0 1.0
DEGDMA
20 l0 5 2.5 1.3 0.6
2.7_+0.12 2.1 ± 0.07 1.5 _+ 0.06 1.3 _+ 0.04 1.2 ± 0.01 1.1 + 0.02
0.1 0.3 0.5 0.7 0.8 0.9
DIPA
l0 5 2.5 1.3 0.6 0.3
1.2 _+ 0.01 1.0 ± 0.06
<0.1 <0.1 <0.1 <0.1 0.7 0.9
DMAPE
DMBZ
40 20 10 5 2.5 1.3 0.6 l0 5 2.5 1.3 0.6 0.3 0.2 0.1
(+)
0.4 0.6 0.8 0.8 1.0 1.0
1.6+0.13 1.5 ± 0.09 1.2+0.12 1.1 + 0.01 1.0 ___0.03 1.1 ± 0.02 1.9 + 0.06
0.1 0.1 0.1 0.4 0.5 0.7 0.8
1.4 1.1 1.0 1.0 1.0
Cell count
Test result +
23 ± 4 76 _ 15 80 _+ 2 102 ± 16 103 ± 17 105 ± 10 111+8 104 + 9
80 96 105 88 108 112 100 91
92 ± 88 ± 79_+ 66 + 105 ± 80_+
5 9 12 13 15 11
77 102 115 128 105 123
56 ± 16 100 ± 16 94 ± 11 91 + 9 105 _+ 10 108 + 6
58 64 99 89 88 96
0 82 + 31 114+ 19 102 ___28 97 + 13
21 62 70 71 84
128 _ 46 114+2 113+ 1 111 + 9 139 + 7
32 67 71 80 79 86
(+)
(+) + 0.06 ± 0.06 + 0.03 + 0.05 _+ 0.03 ± 0.02
2.1
% DNA synthesis
120
+
3
(+) 31+1 51+3 78+7 119±4 115-t-8 128+9
13 14 31 67 81 71
J. Heil et al. / Mutation Research 368 (1996) 181-194
186 T a b l e 2 (continued) Substance
HEMA
TPSb
UDMA
Conc.[mM]
DIT
umu-test Induction rate
G r o w t h factor
4 0
-
-
20
10 5 2.5 1.3 0.6 0.3 0.2
1.3 ± 0.03 1.2 ± 0.02 1.1 ± 0.05 1.0 ± 0.02 1.1 ± 0.03 1.0 ± 0.03 1.0 ± 0.01 1.0 ± 0.02
0.8 0.8 0.9 0.9 0.9 0.9 1.0 0.9
150 75 38 19 10 5 2.5 1.3 0.6 0.3 0.2
0 O. 1 ± 0.02 O. 1 ± 0.02 0.2 ± 0.02 0.4 ± 0.00 0.8 ± 0.08 1.0 ± 0.03 1.1 ± 0 . 1 3 -
0.9 1.1 1.3
% DNA synthesis
Cell count
117_+7 100 ± 5 91 ± 2 97 ± 6 115 _+ 10 102 ± 10 96_+4 96 ± 4
88 102 107 98 104 110 110 97
T e s t result
+
1.1
0.07 0.4 0.5 0.6 -
0.1
-
-
0.04
-
-
6 3 1.5 0.8 0.4 0.2
0.6 0.5 0.6 0.8 0.8 0.8
0.1
-
± + ± ± ± ±
T e s t result
0.10 0.05 0.08 0.06 0.05 0.04
1.5 1.6 1.3 I. 1 1.1 1.1 -
pies are scored as 'limited positive'. A concentration-related increase in umu gene induction strongly supports the assumption of a sample's genotoxicity. 2.4. Cell culture test 2.4.1. D N A synthesis inhibition test (DIT)
The DNA synthesis inhibition test is based on the concept that DNA damage causes inhibition of replicative DNA synthesis that becomes detectable some time after replicating cells have been in contact with genotoxic agents (Painter, 1977; Painter and Howard, 1978). After setting an initial template damage, the rate of DNA synthesis further decreases with time. On the other hand, substances causing nonDNA, cellular alterations, e.g., inhibition of
6±1
13± 1 15± 1 22 ± 1 39 ± 5 54 ± 4 87 _+ 6 1 1 8 + 11 61 56 69 77 57 79 99
± 8 _+ 1 + 1 + 7 _+ I _+ 3 + I
22 44 46 55 67 80 80 100 58 57 46 54 55 50 77
metabolic chains, will be effective only as long as they are in contact with the cells. Once they are removed, the rate of DNA synthesis will increase again in the surviving cell populations. HeLa $3 cells (ATCC, Rockville, MD, USA) were routinely cultured in Eagle's Minimal Essential Medium (MEM without thymidine) supplemented with 10% fetal calf serum, 20 mM HEPES and 100 mg/1 kanamycin in an incubator at 37°C. For use in the DIT (Heil et al., 1990; Heil et al., 1991; Hell and Reifferscheid, 1992), a culture of logarithmically growing HeLa $3 cells was transferred into a single cell suspension by gently detaching the cells with EDTA (250 rag/1 PBS). Then the cells were seeded into 96-well microplates at a densitiy of 2 × 1 0 4 cells/well. The next day, the mono-
J. Heil et al./Mutation Research 368 (1996) 181-194
187
Table 3
Umu test and D I T g e n o t o x i c i t y results o f extracts o f dental m a t e r i a l s (for details, see Section 2: Materials umu-test
Substance Dilution factor
I n d u c t i o n rate
and methods)
DIT G r o w t h factor
T e s t result
% D N A sYnthesis
Cell count
T e s t result
Controls Negative (solvent)
-
1.0 ± 0.03
1.0
100 ± 8
100
Positive ( N Q O )
0.0003 m M
3.0 ± 0.10
1.0
50 ± 6
90
AH26 ~
:1
-
-
M e d i u m eluate
:2 :4 :8
-
<0.1 <0.1
0 7_+2 30+5
30 77 84
: 16 : 32
12.3 ± 1.30
< 0.1 0.3
68 ± 7 76 + 13
89 95
: 64 :128 :256 :512
2.9 _+ 0.10 1.4±0.02 1.1 ± 0 . 0 1 1.0 ± 0.07
0.9 1.0 1.0 1.0
90 _+ 19 119±6 -
106 113 -
+
46
0
AH26 ~
:80
-
-
D M S O eluate
: 160
-
-
: 320 : 640 :1280 :2560 :5120 : 10240 : 20480 : 40960
2.0 ± 0.24 1.4 _+ 0.07 1.1 ± 0.04
<0.1 < 0.1 <0.1 0.4 0.8 0.9
1.0 _+ 0.07
h0
:1
.1 ± 0.03
1.1
121 + 15
83
:2 :4 :8
.1 + 0 . 0 2 .0 _+ 0.06 .3 _+ 0.21
1.2 1.1 0.8
95 -t-7 92 _+ 1 87 ± 5
83 81 83
: 16 : 32 :64
.0 + 0.14 .0 ± 0.03 .0 ± 0.09
1.1 1.1 1.1
113 + 16 120 ± 5 101 ± 4
102 96 101
:128
.1 ± 0 . 5 3
0.9
: 40
1.0 ± 0.01
1.0
: 80 : 160 : 320 :640 : 1280 :2560 :5120
1.2 ± 0.17 1.1 _+ 0.08 1.2 ± 0.11 1.3 ± 0.12 1.4 ± 0.14 1.1 ± 0.08 1.2±09.15
0.9 0.9 0.9 0.9 0.8 0.9 0.9
:81920 Apexit ~ medium
eluate
Apexit a D M S O eluate
(+)
layers of the HeLa cells were exposed for 90 min to the materials to be tested. All concentrations were tested in triplicate; with each set of experiments usually repeated three times.
-
0
22
45 ± 3 74 _+ 2 1 0 2 + 10 115 _ 5 121 _+5 92 _+ 8 -
58 80 93 99 95 98 -
-
-
96± -
l
25 ± 2
16
50 ± 1 71 ± 1 93 ± 1 97 ± 1 102 ± 7 122 ± 7 126_+9
+
+
94
(+)
28 55 63 69 94 90 92
Thereafter, the cells were washed by two rinses with fresh, pre-warmed medium and allowed to recover for 2 h. This was followed by addition of BrdU in a final concentration of 20 ~ M for 60 min.
J. H eil et al. / Mutation Research 368 (1996) 181-194
188 Table 3 (continued)
umu-test
Substance
N2 m e d i u m eluate
Induction rate
G r o w t h factor
Test result
1:1 1:2 1:4 1:8 I : 16 1:32
-
<0.1
(+)
3.5+0.16 1.4 ___0.02 1.2 +- 0.02 1.0 + 0.04 1.0 + 0.02 1.0 +- 0.01 1.0 +- 0.03
1:64
1 : 128
N2 D M S O eluate
Herculite ~ m e d i u m eluate
:40 :80 : 160 : 320 : 640 : 1280 :2560 :5120
1.2 1.1 1.1 1.0 1.0 0.9
1:1 1:2 1:4 1:8 1:16 1 : 32 1 : 64 1 : 128
Herculite ~ D M S O eluate
Sono-CEM ® m e d i u m eluate
DIT
Dilution factor
: 40 : 80 : 160 : 320 :640 : 1280 : 2560 :5120 : 10240 1: 1 1:2 1:4 1:8 1:16 1:32 1 : 64 1:128
_+ 0.06 +- 0.04 +- 0.02 -+ 0.02 + 0.01 _+__0.03
0.1 0.7 0.8 1.0 h0 1.0 1.0 < 0.1 <0.1 0.7 1.0 0.9 1.0 1.1 1.2
1.1 +_ 0.03 1.0 ± 0.05 1.1 _+ 0.04 1.4 _+ 0.30 1.1_+0.02 1.0 +- 0.05 1.0 ± 0.06 1.0_+ 0.14
1.3 1.3 1.2 1.0 1.3 1.2 1.2 1.0
1.2 1.2 1.2 1.4 1.2 1.2 1.2 1.2
1.0 1.0 1.0 1.0 0.8 0.9 0.9 1.0
_+ 0.05 +- 0.03 ± 0.03 _+ 0.34 _+ 0.15 +- 0.18 _+ 0.08 _+ 0.12
-
-
1.0 1.0 1.1 1.1 1.1 1.1 1.0 1.1
+ 0.02 + 0.01 +- 0.11 +0.03 +0.03 ±0.10 + 0.06 +0.19
1.3 1.2 1.1 0.8 1.2 1.2 1.1 0.9
Subsequently, the c e l l s w e r e f i x e d with e t h a n o l / a c e t i c a c i d / w a t e r ( 9 0 : 5 " 5 ) for 30 min at room temperature. The alcohol was poured off and 4 N HCI was added to the fixed cells for 10 rain to denature the DNA. Excess acid was washed away by
% D N A synthesis 0
0 61 56 107 103 118 88
-+ 3 +- 19 -+ 3 + 7 +- 18 +- 26
Cell count
Test result
44 35 48 90 100 113 109 102
+
+ 42 _+ 3 61 _+2 68 + 1 63 +- 3 113±3 102 +- 3 94 _+ 4
60 78 92 98 94 95 103
90+-26
Ill
115 _+ 16
119
107 +- 4 118 +- 27 95 _+ 6 120_+ 11 112+-6 132 +- 23
126 99 105 111 116 92
118_+ 1 108 _+ 6 110+4 108 +- 8 132+--8 112+-4 119+-5 103 +- 15
86 80 80 79 91 86 87 84
1t7--+ 12 106 +_ 2 113+7 134 _+ 7 1 1 6 ± 16 94_+ 10 101 _+ 4 123 + 9
84 98 104 84 99 111 113 94
rinsing the microplate twice with tap water. Then a 1 : 1500 dilution of a monoclonal anti-BrdU antibody (Bio Cell Consulting, Reinach, Switzerland) was added to the cells for 30 rain. After washing the cells three times with tap water, a 1 "500 dilution of
J. Heil et al. / Mutation Research 368 (1996) 181-194
189
Table 3 (continued)
umu-test
Substance Dilution factor Sono-CEM ~' DMSO eluate
Ionoseal ~ medium eluate
Vitrebond ~' medium eluate
DIT Growth factor
: 40 :80 : 160 : 320 : 640 :1280 : 2560 :5120
0.9 _+ 0.01 1.4 ± 0.14 1.2 _+ 0.13 1.3 _+ 0.19 1.2 ± 0.11 1.4+0.18 1.3 ___0.08 1.2 _+ 0.15
0.8 0.8 0.9 0.9 0.8 0.8 0.8 0.9
:1 :2 :4 :8 : 16 : 32 : 64 : 128
0.9+0.11 0.9 -t- 0.08 0.9 + 0.05 0.9-t-0.10 1.0 + 0.08 1.0 _+ 0.02 1.0 + 0.02 0.9 ± 0.07
1.2 1.2 1.1 1.1 1.2 1.1 1.1 1.1
:1 :2
:4 :8 : 16 :32 :64 : 128 : 256 :512 Vitrebond ~ DMSO eluate
Induction rate
1:40
1 : 80 1 : 160 1 : 320 1 : 640 1:1280 1 : 2560 1:5120 1:10240 1 : 20480
-
-
-
-
Test result
%DNA synthesis
Cell count
-
-
18 + 8 43 _+ 6 84 +_ 2 96 _+ 2
5.9_+ 1.12 6.0 _+ 0.67 4.7 _+ 0.06 4.1 +0.12 3.3 _+ 0.12 2.3 + 0.08 2.0 _+ 0.03 1.6+0.10
0.2 0.4 0.6 0.8 0.9 0.9 0.9 0.9
6.2 _+ 0.21 4.5 _+ 0.24 3.1 _+ 0.23 2.0 _+ 0.23 1.7 _+ 0.22 1.4+0.10 1.2 _+ 0.04 1.1 -t- 0.04 1.1 + 0.04 1.1 _+ 0.07
0.5 0.6 0.7 0.9 1.0 0.9 1.0 1.1 1.0 1.0
53 68 78 78
-
+
Test result
-
103 _+ 15 96 _+ 7 89 _+ 6 88 + 3 121 _+ 11 111 _+ 13 119+8 109 + 3
84 83 83 80 97 97 93 90
0 0 3_+3 42 + 3 92 _+ 3 87 + 10 90_+8 99 _+ 16
59 76 94 85 101 115 119 95
123 _+ 10
90 97 97 88 111 104 99 84
110+2 109 _ 2 115+8 123 ___ 17 126 + 3 116_+ 10 98 _+ 12
peroxidase-conjugated F(ab)2-sheep-anti-mouse IgG
e l u t i o n o f the d y e w i t h T r i s b u f f e r a n d c o l o r i m e t r i c
a n t i b o d y ( S i g m a ) w a s a d d e d f o r a n o t h e r 30 m i n . T h e
measurement
cells were w a s h e d three times with tap water, and a
standard
freshly prepared peroxidase substrate solution was
o x i d e ) w a s u s e d as p o s i t i v e c o n t r o l .
added. The color development stop solution (H2804).
at 5 6 4
genotoxin
nm.
I n all e x p e r i m e n t s ,
4-NQO
(4-nitroquinoline
the N-
was stopped with a
T h e e x t i n c t i o n o f the w e l l s
w a s m e a s u r e d at 4 9 5 n m u s i n g a n E L I S A
reader.
2.4.2. Rating o f D I T test results The OPD/SRB
r a t i o s f o r d r u g - t r e a t e d c e l l s are
Cell c o u n t s w e r e d e t e r m i n e d b y s u l f o r h o d a m i n e B
expressed
( S R B ) a d s o r p t i o n to total cell p r o t e i n , f o l l o w e d b y
i n t o D N A . T h e r e s u l t s o f t h e t e s t s a r e g i v e n as m e a n
as p e r c e n t a g e s
of control
incorporation
190
J. Heil et al. / Mutation Research 368 (1996) 181-194
of triplicate determinations _+ standard deviation. If a Dis0 value (cell count corrected DNA synthesis of 50%, as compared to untreated controls) could be derived, the carcinogen-induced DNA synthesis inhibition is considered 'positive'. Samples showing no resp. moderate signs of concomitant cytotoxicity (cell count > 40%) are scored 'sufficient positive', the presence of higher degrees of cytotoxicity (cell count < 40%) sets scoring to 'limited positive'.
2.5. bl vivo-assay
recovered by shaking them in 5 ml of elution buffer for 2 h, using an Eppendorf shaker (1500 U/min). Aliquots of 0.2 ml were taken for the fluorometric
Table 4 A F E (in v i v o alkaline filter elution t e c h n i q u e ) g e n o t o x i c i t y results o f c h e m i c a l m o n o s u b s t a n c e s present in dental materials and extracts o f dental materials (for details, see Section 2: Materials and methods) Substance
2.5.1. Alkaline elution technique The alkaline elution technique assays for DNA strand breaks which are the most frequent consequence of exposure of cells or live animals to DNA damaging substances. Filter elution was performed according to the procedure of Kohn et al. (1976), which had been adapted to gills of freshwater clams Dreissena polymorpha and Corbicula fluminea by Waldmann et al. (1995). The use of clams or other lower invertebrates avoids experimental restrictions given by official directions. In alkaline elution experiments, clams (18-20 mm long, weighing 2.2 to 2.5 g) were exposed in 200 ml of 'low-salt water' supplemented with the extracts of dental materials. Circulation of water during exposure was provided by magnetic stirrers. Four specimens of clams per assay were incubated for 2 h in light-protected 500-ml glass beakers. After exposure, the gills of the clams were removed, immediately homogenized and the homogenate was transferred onto a filter apparatus for alkaline elution analysis. Aliquots from the homogenate were layered onto 0.22-/xm pore size polyvinyl fluoride filters (25 mm, Millipore), washed with 5 ml ice-cold PBS and then lysed by percolating 4 ml of a solution containing 2 M NaCI, 0.3% sodium lauroyl sarkosine, 0.02 M EDTA and 0.5 m g / m l proteinase-K (pH 8.7). The lysed homogenate residues on the filter were washed with 10 ml of a 0.02 M EDTA solution (pH 8.7). This procedure was followed by alkaline elution with tetraethylammoniumhydroxide buffer (pH 12.4) and 0.02 M EDTA. The flow rate of the elution was 0.04 ml/min; six 1.8 ml fractions were collected per channel. After elution, the DNA from the filters was
Concentration
A F E factor
[mM]
2-h incubation
24-h incubation
Test result
BHT Bis-GMA CQ CSA DCHA
2 2 2 2 2
1.00 1.07 1.15 1.10 1.25
(+ )
DMAPE DMBZ HEMA
2 2 2
0.92 1.27 1.0
(+ ) -
TPSb UDMA
6 2
O.7 1.24
+ (+ )
Dilution factor AH26 ~
1:40
1.84
-
( m e d i u m extract)
1:80 1 : 2500
1.45 -
1.32
A H 2 6 ~' ( D M S O extract)
1 : 60
1.31
Apexit ~ ( m e d i u m extract)
1 : 27 1 : 118
1.05 0.84
N2
1 : 143
0.88
Herculite ~ ( m e d i u m extract)
l:27
1.02
Sono-CEM * ( m e d i u m extract)
1:27
0.96
Vitrebond e ( m e d i u m extract)
1 : 27 1:50 1 : 67
1.67 1.38 1.06
-
1 : 1000
-
1 : 2500
-
1.33 1.09
1:1000
-
1.24
( m e d i u m extract)
Vitrebond ~ ( D M S O extract)
+
-
-
191
J. Hell et al, /Mutation Research 368 (1996) 181-194
D N A determinations of the D N A eluted in the fractions. Fluorescence was measured by an automated version of the procedure described by Stout and Becker (1982).
3. Results Tables 2 - 4 survey the results of the genotoxicity testing of 14 chemical monosubstances present in dental materials and 7 extracts. It's striking that most agents tested yield 'positive' results in at least one of the genotoxicity tests; however, with effects ranging from 'borderline' to 'strong positive'. The higher percentage of 'positives' in the eucaryotic DIT, to a certain degree, can be attributed to a concomitant high toxicity of many compounds which kills most tester bacteria and counteracts positive outcomes in bacterial genotoxicity/mutagenicity tests. Umu growth factor values > '1' indicate that such sub-
2.5.2. Rating of afe test results The slope factors of the D N A alkaline elution curves, indicating the number of single-strand breaks in the D N A from the treated animals, in relation to their appropriate controls, are expressed as arbitrary units ('AFE-factor'). A factor > 1.3 is used as an indicator for a 'sufficient positive' genotoxic potential of a substance ('limited positive' = factor > 1.2). Slope factors < 0.7 are indicative of D N A - D N A crosslinks and scored as 'sufficient positive'.
--=--
f
induction rate
....... Vo,,~ h M d o r
- - = - - induction rate ......... grow~l ~ ' o r
5
• 1,2 14-
- 1,4
T 12-
AH26
10-
......
medium
~
e~tmct
/
umu
-1.o
4-
- 1,2 umu
-0,8
- 0,6
g -
~-~
-1.0
Herculite medium ¢dract
,0.6 ~
- 0,4 ~
• 4-
.0,4 1-
-0,2
20 0,001
i
. . . . . . . .
. . . . . . . .
• 0.2
0,0
0,01
. . . . . . . .
0 • ' '0.01
0,1
. . . . . . .
i 1
01,1
dilution factor
dilution factor
- - = - - D N A syndlesi$ cell count
1
--I--
......
J
~2o~
140-
100 i
~2o-"
T •
........
~
.....
DNA s~thesis cell count
T DIT
.
.m 100 i
"~ 80 O~ 60-
'3£\
7< 4oO A1-126
2o~
medium ~dmet
01 ....
i
0,01
\,
. . . . . . . .
i
0,1
dilution factor
r i O.O
. . . . . . . .
.~,
80-
Z a
60-
•
4o-
-
20-"
i
1
O"
Herculite m e d i u m eoctm~
........
, ,,,i
;
dilution factor
Fig. 1. Effects of the medium extracts of AH26 ~ and Herculite '~ in u m u - t e s t and D N A synthesis inhibition test (DIT).
1
I
J
192
J. Heil et al. /Mutation Research 368 (1996) 181-194
I
--m-- inductionrate1 growth~¢torI
--=-- DNAsyn~esis cell count
. . . . . . . .
1.2 120-
1,oo
DIT
100'~ 00.
1 0,4 0,6
~
60-
z
4o.
\,
2-
Vitrebond me01umextract
0,2 0 0.001
.
.
.
.
.
.
.
0.~1
.......
0'.1
•
0.0
dilution factor
....
i
o,01
. . . . . . . .
i
o,1 dilution factor
Fig. 2. Effect of the medium extract of Vitrebond ~ in umu-test and DNA synthesis inhibition test (DIT).
stances (Bis-EMA, Bis-GMA, UDMA) are bonding agents which make scoring more difficult. In the case of the widely used dental materials AH26 ® and Vitrebond ®, the in vivo - tests with the alkaline elution method confirmed the in vivo - relevance of 'strong positive' in vitro results.
4. Discussion Genotoxlcity is one of the many types of adverse effects of chemicals. Since most dental materials release small amounts of various substances into their physiological environment (pulp, oral cavity), appropiate regulations have to ensure that the concern about the (possible) genotoxicity/mutagenicity/carcinogenicity of dental materials is abolished or at least minimized. The problem connected herewith is less the analytical determination of a substance but more "what is the concentration of the substance that causes harm?". The recent European Standard "Biological evaluation of medical devices, Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicity" (EN 30993-3, ISO 10993-3:1992) recommends methods which are listed in the "OECD Guidelines For The Testing Of Chemicals". For certain aspects, alternative methods can be accepted. Nevertheless, screening tests should be performed 'state of the art', i.e., tests using microplates should be preferred, allowing for the fast, easy and simultaneous testing of extensive numbers of data points. Since many dental
materials are highly toxic (Leinenbach et al., 1993; Karbakhsch et al., 1994), it is a basic requirement for genotoxicity tests to easily quantify simultaneous cytotoxicity in order to avoid misinterpretation of the data. The umu-test and the DNA synthesis inhibition test both fulfil this criterion, whereas, e.g., Ames test results are usually published without adequate toxicity data. Moreover, it cannot be excluded that socalled 'small colonies' (i.e., survivors of the toxic effect of the test material which grow by using up the histidine in the top agar) are counted as revertants (Stea et al., 1994). The root canal filling material AH26 ® has already been reported to be mutagenic in the Ames test. These findings could be confirmed by strong genotoxic responses in the bacterial umu-test (Fig. 1), the eucaryotic DIT (Fig. 1) and the in vivo induction of DNA fragmentation, as measured by the alkaline filter elution technique (Table 4), indicating that the DNA-damaging properties of AH26 ® have to be taken serious. It is remarkable that in the umu-test AH26 ® features genotoxic responses up to a 10000-fold dilution when extracted with DMSO, whereas the medium extract is less genotoxic by a factor of about 100. These findings are equivalent to Stea et al. (1994) and Schweikl et al. (1994) who report the mutagenicity of DMSO extracts of AH26 ® to Salmonella typhimurium. In contrast to Schweikl et al. (1994) but in accordance with Stea et al. (1994) is the observation that mutagenic substances of AH26 ® (presumably an epoxy-derivative of bisphenol-(A)-diglycidylether) are present even in the poly-
z Heil et al./Mutation Research 368 (1996) 181-194
merized material. This supports the hypothesis that mutagenic agents may migrate into the surrounding tissue. Although human exposure will be maximal in the first hours after implementation, a prolonged exposition time may drastically reduce the concentrations required for significant genotoxic effects, as shown in the AFE (Table 4). Vitrebond ® is a glass ionomer cement which elicits strong genotoxic responses in all three test systems (Fig. 2, Table 4). In the u m u - t e s t , the least significant dilution of the extract is about 1000fold in both the DMSO and medium extract. In the DIT, however, only the extract with physiological cell culture medium caused effects indicative of genotoxic activity, whereas the DMSO extract didn't lead to a significant inhibition of DNA synthesis, presumably because of it's higher dilution due to the toxic effect of DMSO itself. In contrast to AH26 ®, the genotoxic effects of Vitrebond ® are nearly unaffected by accompanying cytotoxic effects. In the AFE, a prolonged exposition time (24 h vs. 2 h) reduced the concentrations required for significant genotoxic effects by a factor of approx. 20 (Table 4). Zinc-Eugenol cements like N2 ® are used in general dental practice as a filling of root canals. Eugenol (4-allyl-2-methoxyphenol, a natural substance from the oil of cloves) was previously found to behave as a genotoxin in several in vitro systems including the Ames test (RTECS, 1995). In DIT and u m u - t e s t , extracts of N2 ® produced effects indicative of genotoxic activity, too. Whether Eugenol or another substance is the causative agent for this, remains to be established. CQ is a substance which is sufficient positive in both the u m u - t e s t and the DIT. There is no concomitant cytotoxicity which could interfere with the genotoxicity measurements and even the effective concentrations are in the same range. CSA, a derivative of CQ, is sufficient positive in the DIT, but since the u m u induction rates > 1.5 are accompanied with growth factors < 0.5, the u m u effects have to be taken with caution. TPSb is negative in the u m u - t e s t . Higher concentrations ( > 10 mM) lead to a complete inhibition of procaryotic as well as eucaryotic cell metabolism. In the DIT, TPSb shows a 'classic' concentration-inhibition curve, ranging from 5 mM to 0.04 mM. Hence, TPSb has to be classified as sufficient posi-
193
tive in the DNA synthesis inhibition test. Furthermore, the result of the in vivo - alkaline elution assay supports the hypothesis that TPSb may possess a genotoxic potential, presumably by causing (proteinase-K resistant) DNA-DNA crosslinks. Herculite ® is an example for a dental composite which gave negative results in both DIT and u m u - t e s t (Fig. 1). Interestingly, Herculite ®, according to manufacturer's description a composition of 16 components, contains substances which have been tested 'positive' in DIT a n d / o r u m u - t e s t (Bis-EMA, BisGMA, CQ, CSA, DEGDMA, TPSb). The reason for this discrepancy in the outcome of the tests certainly is complex ('modulation'), but it cannot be excluded that simple concentration effects contribute to this phenomenon. HEMA, which has wide applicability in medical technology, was not genotoxic under the conditions of the three bioassays used. The apparent non-genotoxicity of some substances, however, may be due to insufficient metabolic activation by the tester organisms. Nevertheless, additional tests with a liver microsomal activation system ($9 mix) were not performed, since the dental materials investigated in this study do not contain molecular structures which could be activated by $9 mix. Furthermore, pre-studies have given evidence that the presence of $9 reduces effects (data not shown). Non-genotoxic carcinogens, many of them presenting strong cytotoxic properties, induce tumors via an indirect threshold mechanism. Since the overstimulation of cell division (mitogenesis) enhances tumor production (Ames and Gold, 1990), the continued irritation of the oral tissues adjacent to certain dental materials may be seen as an additional risk factor. Of course, this does not automatically involve serious health consequences, but reducible hazards should be eliminated. The point in question normally involved with in vitro-tests is their extrapolation to any human risk connected therewith. Many mathematical models have been advanced to carry out the extrapolation from in vitro results to small doses in vivo, but most assumptions on the hazards from small amounts of carcinogens are unverifiable. Taken together, the results of this study give support to considerations that besides their benefits several dental materials possess unwanted properties
194
J. Heil et al. /Mutation Research 368 (1996) 181-194
that can be a threat to human health. Fortunately, there are safer alternatives available, particularly when one bears in mind that many composites are not only interchangeable, but made out of the same set of monosubstances. In the long run, final biological evaluation of dental materials has to be done by a group of experts on the basis of published results and adequate knowledge.
Acknowledgements This stuty was supported by a grant of the DFG (Ge 455/4-2).
References Ames, B.N. and L.S. Gold (1990) Too many rodent carcinogens: mitogenesis increases mutagenesis, Science, 249, 970-971. Battista, J.R., T. Ohta, T. Nohmi, W. Sun and G.C. Walker (1990) Dominant negative umuD mutations decreasing RecA-mediated cleavage suggest roles for intact UmuD in modulation of SOS mutagenesis, Proc. Natl. Acad. Sci. USA, 87, 7190-7194. Heft, J. and G. Reifferscheid (1992) Detection of mammalian carcinogens with an immunological DNA-synthesis inhibition test, Carcinogenesis, 13, 2389-2394. Heil, J., G. Reifferscheid, E. Pischtschan, M. Kl~is, H.-D. Jungbluth and R.K. Zahn (1990) A microplate version of the DNA-synthesis inhibition test et al.A microplate version of the DNA-synthesis inhibition test (DIT) for rapid detection of DNA-alteration potentials, Anal. Biochem., 187, 314-317. Heil, J., G. Reifferscheid, D. Hellmich, M. Hergenr/ider and R.K. Zahn (1991) Genotoxicity of the fungicide Dichlofluanid in seven assays, Environ. Mol. Mutagen., 17, 20-26. Karbakhsch, M., G. Leyhausen and W. Geurtsen (1994) Biocompatibility of light curing glass ionomer cements in various cell cultures, J. Dent. Res., 73, 183. Kohn, K.W., L.C. Erickson, R.A.G. Ewing and C.A. Friedman (1976) Fractionation of DNA from mammalian cells by alkaline elution, Biochemistry, 15, 4629-4637. Lehmann, F., G. Leyhausen and W. Geurtsen (1993) Cytotoxic alterations in different fibroblast cultures caused by matrix monomers, J. Dent. Res., 72, 219.
Leinenbach, F., G. Leyhausen and W. Geurtsen (1993) Biocompatibility of root-canal filling materials in fibroblast cultures, J. Dent. Res., 72, 306. Leyhausen, G., J. Heil, G. Reifferscheid and W. Geurtsen (1995) Das gentoxische Potential yon Kompositbestandteilen, Dtsch. Zahn~irztl. Z., 50, 134-136. McDaniels, A.E., A.L. Reyes, L.J. Wymer, C.C. Rankin and G.N. Stelma, Jr. (1990) Comparison of the Salmonella (Ames) Test, Umu Tests, and the SOS Chromotests for Detecting Genotoxins, Environ. Mol. Mutagenesis, 16, 204-215. Oda, Y., S. Nakamura, I. Oki, T. Kato and H. Shinagawa (1985) Evaluation of the new system (umu-test) for the detection of environmental mutagens and carcinogens, Mutation Res., 147, 219-229. Painter, R.B. (1977) Rapid test to detect agents that damage human DNA, Nature, 265, 650-651. Painter, R.B. and R. Howard (1978) A comparison of the HeLa DNA-synthesis inhibition test and the Ames test for screening of mutagenic carcinogens, Mutation Res., 54, 113-115. Reifferscheid, G., J. Heft, Y. Oda and R.K. Zahn (1991) A microplate-version of the SOS/umu-test for rapid detection of genotoxines and genotoxic potentials of environmental sampies, Mutation Res., 253, 215-222. RTECS (Registry of Toxic Effects of Chemicals); data taken from Chem-Bank ~ CD-ROM (May 1995), SilverPlatter Information, Inc., Norwood, MA, USA. Schweikl, H., G. Schmalz and B. Bey (1994) Mutagenicity of dentin bonding agents, J. Biomed. Materials Res., 28, 10611067. Spahl, W. and H. Budzikiewicz (1994) Qualitative analysis of dental resin composites by g a s a n d liquid chromatography/mass spectrometry, Fresenius J. Anal. Chem., 350, 684-691. Spahl, W., H. Budzikiewicz and W. Geurtsen (1991) Eine Untersuchung zum Restmonomer- und Additivagehalt verschiedener lichtNirtender Hybridkomposite, Dtsch. Zahn~irztl. Z., 46, 471-473. Stout, D.L. and Becker, F.F. (1982)Anal. Biochem. 187, 302-307. Stea, Su., L. Savarino, G. Ciapetti, E. Cenni, St. Stea, F. Trotta, G. Morozzi and A. Pizzoferrato (1994) Mutagenic potential of root canal sealers: Evaluation through Ames testing, J. Biomed. Material Res., 28, 319-328. Waldmann, P., B. Pivcevic, W.E.G. MUller, R.K. Zahn and B. Kurelec (1995) Increased genotoxicity of acetylaminofluorene by modulators of multixenobiotic resistance mechanisms: Studies with the fresh water clam Corbicula fluminea, Mutation Res., 342, 113-123.
ELSEVIER
Mutation Research 368 (1996) 181- 194
Genotoxicity of dental materials JiJrgen Heil a,*, Georg Reifferscheid a, Petra Waldmann a, Gabriele Leyhausen Werner Geurtsen b
b,
a AMMUG, Universit~it Mainz, Obere Zahlbacher Str. 63, D-55101 Mainz, Germany h Poliklinikfiir Zahnerhaltung und Parodontologie. Medizinische Hochschule Hannol,er, Konstanty-Gutschow-Str. 8, D-30625 Hannoz,er, Germany
Received 8 January 1996; revised 1 February 1996;accepted 2 February 1996
Abstract This study was performed to characterize the (possible) DNA-damaging properties of dental materials and to identify specific compounds that contribute to this genotoxicity. For screening, three tests that assay for different aspects of genotoxicity (i) the bacterial umu-test; (ii) the eucaryotic DNA synthesis inhibition test; and (iii) the in vivo alkaline filter elution technique were chosen. This investigation gives several lines of evidence that most dental materials tested (14 chemical monosubstances present in dental devices and 7 extracts of dental materials) yield 'positive' results in at least one of the genotoxicity tests, however, with effects ranging from 'borderline' to 'strong positive'. The extracts of the widely used dental materials Vitrebond® and AH26 ® elicited clear concentration-related genotoxic responses in all test systems. On the basis of these data and public concern, more attention has to be given to local or systemic complications which may be associated with the use of dental materials. Kevwords: Genotoxicity;Dental material; Umu-test; DNA synthesis inhibition;Alkaline elution technique
1. Introduction Since 1995 dental materials are classified as medical products. Therefore, it is indispensable according to national as well as international regulations that medical devices - either newly developed or currently in use - have been proven for biocompatibility and that their toxicological profile has been determined. Previous studies have shown that dental materials may induce local and systemic adverse effects which are caused by extractable monomers
* Corresponding author. Tel.: +49-6131-173314; Fax: +496131-173364
(Spahl and Budzikiewicz, 1994; Spahl et al., 1991) a n d / o r other inorganic and organic ingredients. Various in vivo and in vitro test systems have been used in order to identify the components responsible for the reduction of the biocompatibility of these materials (Lehmann et al., 1993). Meanwhile, there are some few papers dealing with the genotoxic activity of selected dental materials (Leyhausen et al., 1995; Schweikl et al., 1994; Stea et al., 1994), mostly Ames test data. Out of 12 commercially available dental cements, Stea et al. found one cement clearly mutagenic to Salmonella typhimurium; however, with nearly all cements showing strong bactericidal activity. This fact, among
0165-1218/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S0165-1218(96)00011-0
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J. Heil et al. /Mutation Research 368 (1996) 181 194
others, indicates that bacterial test systems cannot be the only basis for the assessment of the DNA-damaging properties of medical products. This study, which came into being by a close cooperation of genetic toxicologists with dentists, reports the outcome of genotoxicity studies of some widely used dental materials and their components. To our knowledge, this is the first compilation of results obtained with three test systems covering different steps of mutagenesis/ carcinogenesis: (i) the bacterial umu-test, monitoring the mutagenic potential of a substance; (ii) the eucaryotic DNA synthesis inhibition test, detecting potential carcinogenic substances; and (iii) an in vivo alkaline filter elution technique, sensitive to strand breaks. These special methods - primarily developed by our group for the testing of environmental samples and in successful use for several years - have been combined to a battery of genotoxicity tests which is fast, easy to perform and statistically reliable.
tration in medium was set to 1.2% (cell culture test) resp. 3% (bacterial test).
2. Materials and methods
2.2.2. Extracts of dental materials Dental materials tested are listed in Table lB. These materials often consist of about 10-20 single ingredients, mainly anorganics (e.g., SiO 2, ZnO, Na3AIF6, A1203, Bi203, Ca(OH) 2, Ca3(PO4) 2, TiO 2, YF3), various methacrylates and others (e.g., initiators, inhibitors, color pigments, natural oils). All materials were prepared according to the manufacturer's instructions. Briefly, 300 mg of each root canal filling material (AH26 ®, N2 ®, Apexit ®) were mixed and placed in screwcaps of polyethylene vials and set for 3 days at 37°C and 100% humidity. Composites (Herculite XR ®, Sono-CEM ~) and glass ionomer cements (Ionoseal ®, Vitrebond ®) were mixed and polymerized in silicone molds for 60 s by a blue light source (Elipar II, Espe, Seefeld, Germany). Thereafter, the hardened specimens were extracted with 5 ml of serum- and antibiotics-free cell culture medium or 5 ml of DMSO for 24 h at 37°C. Extracts were stored frozen at - 2 0 ° C until use.
2.1. Materials
2.3. Bacterial test system
Dental materials tested were aliquots from materials used for clinical purposes. Supplies (microplates, cell culture flasks) were from Greiner, Frickenhausen, Germany; cell culture media and sera were obtained from Biochrom (Berlin, Germany). Fine chemicals as well as peroxidase-conjugated antimouse IgG antibody were purchased from Sigma (Deisenhofen, Germany). Monoclonal anti-BrdU antibody was from Bio Cell Consulting (Reinach, Switzerland).
2.3.1. Umu-test The umu-test has been developed for the detection of genotoxins by Oda et al. (1985), who generously provided us with the tester strain. The test is based on the capability of genotoxic agents to induce the umu operon in Salmonella typhimurium strain TA1535/pSKI002. Since the umuC gene in the bacteria is fused with the lacZ gene for/3-galactosidase, the induction rate of umuC can be easily assessed by determination of /3-galactosidase activity. The products of the SOS-regulated umuCD operon are directly involved in both point and frameshift mutagenesis, bacteria lacking these (or functionally analogous) mutator genes can be viewed as nearly nonmutable (Battista et al., 1990). Originating from the (quite insensitive) Ames tester strain TA1535, this (sensitive) umu strain was constructed by introducing the plasmid pSK1002 into the bacteria. Like in the (sensitive) Ames tester strains TA98 and TAI00, which carry an analogous plasmid (pKM101) coding for error-prone DNA repair (SOS mutagene-
2.2. Sample preparation 2.2.1. Monosubstances The chemicals (purity > 98%) are listed in Table 1A. Most substances require the use of an organic solvent to enhance solubilization. Each substance was dissolved in DMSO at a stock concentration of 2 M. This stock solution was serially diluted in 1:2 steps and transferred onto the microplate with the tester organisms using a laboratory workstation (Merlin, Bonn, Germany). The final DMSO concert-
J. Heil et al. /Mutation Research 368 (1996) 181-194
sis), this greatly enhances survival after exposure to genotoxins; however, at the cost of an increased mutation rate (i.e., higher sensitivity of such tester strains). Not least on this account, a recent study confirmed that the results of the umu test are statistically equivalent to the Ames test (McDaniels et ai., 1990). The umu test was performed in microplates as described by Reifferscheid et al. (1991). Salmonella from stock were grown in nutrient broth for the overnight culture. Logarithmically growing tester bacteria were exposed to varying concentrations of the test material. All concentrations were tested in triplicate; with each set of experiments usually repeated three times. After 2 h of exposure, the bacterial suspension was diluted 10-fold, followed by a subsequent additional incubation period of 2 h. Thereafter, bacterial growth was measured as turbid-
183
ity (E600) with a microplate reader. The D N A damage induced expression of u m u C was quantified via the determination of /3-galactosidase activity at 420 nm using O N P G (o-nitrophenyl-/3-mgalactopyranoside; Sigma) as a substrate. In all experiments, the standard genotoxin 4-NQO (4-nitroquinoline Noxide) was used as positive control. 2.3.2. Rating o f umu test results
The results of the tests are given as mean of triplicate determinations +__standard deviation. According to the DIN (Deutsche Industrie Norm) and ISO (International Organization for Standardization) working draft ' u m u test', an induction rate of the umu gene exceeding 1.5-fold control values is scored as 'sufficient positive'. Since it cannot be excluded that concomitant cytotoxicity (growth factor < 0.5) interferes with genotoxicity assessment, these sam-
Table 1 A. List of monosubstances tested Abbreviation
Chemical composition
Function
BEMA BHT Bis-EMA Bis-GMA CQ CSA DCHA DEGDMA DIPA DMAPE DMBZ HEMA TPSb UDMA
benzylmethacrylate butylated Hydroxytoluene ethoxylated bisphenol-(A)-glycidylacrylate bisphenol-(A)-glycidylacrylate campherquinone campheracidanhydride dicyclohexylamine diethyleneglycoldimethacrylate 2,6-dilsopropylaniline 2-(4-dimethylaminophenyl)ethanol 2,2-dimethoxybenzoin 2-hydroxyethylmethacrylate triphenylstibane urethanedimethacrylate
monomer inhibitor monomer monomer photoinitiator derivative of CQ coinitiator monomer coinitiator coinitiator photoinitiator monomer derivative of Bis-GMA monomer
B. List of dental materials tested Abbreviation
Field of application (manufacturer)
AH26°~ APEXCIT ~ N2 ~ HERCULITE* SONO-CEM® IONOSEAL~ VITREBOND~
root canal filling material (De Trey, Germany) root canal filling material (Vivadent, Liechtenstein) root canal filling material (Indrag AGSA, Switzerland) light curing hybrid composite for restorations (Kerr, USA) light curing hybrid composite for restorations (ESPE, Germany) light curing glass ionomer cement for cavity lining (VOCO, Germany) light curing glass ionomer cement for cavity lining (3M, Medica, Germany)
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Table 2 UMU test and DIT genotoxicity results of chemical monosubstances identified in dental materials (for details, see Section 2: Materials and Methods) Substance
Conc.[mM]
umu-test Induction rate
Controls Negative (solvent) Positive (NQO) BEMA
0.0003
DIT Growth factor
1.0 -t- 0.03 3.0 ± 0.10
Test result
% DNA synthesis
Cell count
1.0 1.0
100 ± 8 50 ± 6
100 90
112_+2 99 _+ 7 125 _+ l0 115_+4 98 _+ 7 103 + 2
94 94 94 97 100 94
Test result
5 2.5 1.3 0.6 0.3
1.4 1.3 1.2 1.1
± 0.02 ± 0.03 ± 0.03 +0.04
< 0.1 <0.1 0.5 0.6 0.6 0.8
BHT
6 3 1.5 0.8 0.4
0.8 0.7 0.7 0.8 0.8
± 0.01 ± 0.07 -+ 0.02 -+ 0.03 ± 0.04
1.0 1.1 h1 1.0 1.0
26 ± 5 14±2 20 _+ 6 62 ± 15 97 ± 7
30 23 32 72 96
(+)
Bis-EMA
6 3 1.5 0.8 0.4 0.2
0.5 0.6 0.7 0.6 0.6 0.7
-+ 0.04 ± 0.08 ± 0.05 ± 0.08 ± 0.03 -- 0.07
2.1 2.0 1.6 1.6 1.6 1.5
39 40 60 58 70 86
59 49 49 63 60 52
+
Bis-GMA
150 75 38 19 10 5 2.5 1.3 0.6 0.3 0.15 0.08 0.04 0.02
0.9 0.5 0.5 0.5 0.6 0.6 0.8 0.8 -
± 0.02 ± 0.04 ± 0.02 ± 0.04 ± 0.02 ± 0.05 -+ 0.08 _+ 0.06
1.2 2.0 1.7 1.9 1.8 1.5 1.2 1.2
20 l0 5 2.5 1.3 0.6 0.3 0.2
4.1 3.4 1.7 1.2 1.1 1.0 1.0 1.0
± 0.21 ± 0.06 ± 0.05 _+ 0.02 ± 0.03 ± 0.01 ± 0.02 ± 0.01
CQ
10
-
+
-
0.1 0.3 0.5 0.6 0.8 0.9 0.9 0.9
± 5 ± 1 _+ 4 ± 9 ± 6 ± 5
23 ± 1 19+ 1 18+ 1 50 ± 4 123 _+ 11 105 ± 6
28 46 48 50 53 77
31 + 2 86 ± 2 91 + 5 117___2 116+7 113_+6 105 _+ 9
44 70 91 87 98 96 98
+
+
J. Heil et al. / Mutation Research 368 (1996) 181-194
185
Table 2 (continued) Substance
CSA
Conc.[mM]
20 10 5 2.5 1.3 0.6 0.3 0.2
umu-test
DIT
Induction rate
Growth factor
Test result
-
<0.1
(+)
-
<0.1
3.0±0.07 1.6±0.04 1.3±0.00 1.1±0.03 1.0±0.01 1.0±0.03
0.1 0.4 0.7 0.8 0.8 0.8
0.1
-
-
0.05
-
0.03
-
0.01
-
-
-
DCHA
20 10 5 2.5 1.3 0.6
1.3 1.1 1.0 1.0 1.0 1.0
_+ 0.01 ± 0.04 ± 0.02 _+ 0.02 _+ 0.00 _+ 0.00
0.8 0.8 0.8 0.9 1.0 1.0
DEGDMA
20 l0 5 2.5 1.3 0.6
2.7_+0.12 2.1 ± 0.07 1.5 _+ 0.06 1.3 _+ 0.04 1.2 ± 0.01 1.1 + 0.02
0.1 0.3 0.5 0.7 0.8 0.9
DIPA
l0 5 2.5 1.3 0.6 0.3
1.2 _+ 0.01 1.0 ± 0.06
<0.1 <0.1 <0.1 <0.1 0.7 0.9
DMAPE
DMBZ
40 20 10 5 2.5 1.3 0.6 l0 5 2.5 1.3 0.6 0.3 0.2 0.1
(+)
0.4 0.6 0.8 0.8 1.0 1.0
1.6+0.13 1.5 ± 0.09 1.2+0.12 1.1 + 0.01 1.0 ___0.03 1.1 ± 0.02 1.9 + 0.06
0.1 0.1 0.1 0.4 0.5 0.7 0.8
1.4 1.1 1.0 1.0 1.0
Cell count
Test result +
23 ± 4 76 _ 15 80 _+ 2 102 ± 16 103 ± 17 105 ± 10 111+8 104 + 9
80 96 105 88 108 112 100 91
92 ± 88 ± 79_+ 66 + 105 ± 80_+
5 9 12 13 15 11
77 102 115 128 105 123
56 ± 16 100 ± 16 94 ± 11 91 + 9 105 _+ 10 108 + 6
58 64 99 89 88 96
0 82 + 31 114+ 19 102 ___28 97 + 13
21 62 70 71 84
128 _ 46 114+2 113+ 1 111 + 9 139 + 7
32 67 71 80 79 86
(+)
(+) + 0.06 ± 0.06 + 0.03 + 0.05 _+ 0.03 ± 0.02
2.1
% DNA synthesis
120
+
3
(+) 31+1 51+3 78+7 119±4 115-t-8 128+9
13 14 31 67 81 71
J. Heil et al. / Mutation Research 368 (1996) 181-194
186 T a b l e 2 (continued) Substance
HEMA
TPSb
UDMA
Conc.[mM]
DIT
umu-test Induction rate
G r o w t h factor
4 0
-
-
20
10 5 2.5 1.3 0.6 0.3 0.2
1.3 ± 0.03 1.2 ± 0.02 1.1 ± 0.05 1.0 ± 0.02 1.1 ± 0.03 1.0 ± 0.03 1.0 ± 0.01 1.0 ± 0.02
0.8 0.8 0.9 0.9 0.9 0.9 1.0 0.9
150 75 38 19 10 5 2.5 1.3 0.6 0.3 0.2
0 O. 1 ± 0.02 O. 1 ± 0.02 0.2 ± 0.02 0.4 ± 0.00 0.8 ± 0.08 1.0 ± 0.03 1.1 ± 0 . 1 3 -
0.9 1.1 1.3
% DNA synthesis
Cell count
117_+7 100 ± 5 91 ± 2 97 ± 6 115 _+ 10 102 ± 10 96_+4 96 ± 4
88 102 107 98 104 110 110 97
T e s t result
+
1.1
0.07 0.4 0.5 0.6 -
0.1
-
-
0.04
-
-
6 3 1.5 0.8 0.4 0.2
0.6 0.5 0.6 0.8 0.8 0.8
0.1
-
± + ± ± ± ±
T e s t result
0.10 0.05 0.08 0.06 0.05 0.04
1.5 1.6 1.3 I. 1 1.1 1.1 -
pies are scored as 'limited positive'. A concentration-related increase in umu gene induction strongly supports the assumption of a sample's genotoxicity. 2.4. Cell culture test 2.4.1. D N A synthesis inhibition test (DIT)
The DNA synthesis inhibition test is based on the concept that DNA damage causes inhibition of replicative DNA synthesis that becomes detectable some time after replicating cells have been in contact with genotoxic agents (Painter, 1977; Painter and Howard, 1978). After setting an initial template damage, the rate of DNA synthesis further decreases with time. On the other hand, substances causing nonDNA, cellular alterations, e.g., inhibition of
6±1
13± 1 15± 1 22 ± 1 39 ± 5 54 ± 4 87 _+ 6 1 1 8 + 11 61 56 69 77 57 79 99
± 8 _+ 1 + 1 + 7 _+ I _+ 3 + I
22 44 46 55 67 80 80 100 58 57 46 54 55 50 77
metabolic chains, will be effective only as long as they are in contact with the cells. Once they are removed, the rate of DNA synthesis will increase again in the surviving cell populations. HeLa $3 cells (ATCC, Rockville, MD, USA) were routinely cultured in Eagle's Minimal Essential Medium (MEM without thymidine) supplemented with 10% fetal calf serum, 20 mM HEPES and 100 mg/1 kanamycin in an incubator at 37°C. For use in the DIT (Heil et al., 1990; Heil et al., 1991; Hell and Reifferscheid, 1992), a culture of logarithmically growing HeLa $3 cells was transferred into a single cell suspension by gently detaching the cells with EDTA (250 rag/1 PBS). Then the cells were seeded into 96-well microplates at a densitiy of 2 × 1 0 4 cells/well. The next day, the mono-
J. Heil et al./Mutation Research 368 (1996) 181-194
187
Table 3
Umu test and D I T g e n o t o x i c i t y results o f extracts o f dental m a t e r i a l s (for details, see Section 2: Materials umu-test
Substance Dilution factor
I n d u c t i o n rate
and methods)
DIT G r o w t h factor
T e s t result
% D N A sYnthesis
Cell count
T e s t result
Controls Negative (solvent)
-
1.0 ± 0.03
1.0
100 ± 8
100
Positive ( N Q O )
0.0003 m M
3.0 ± 0.10
1.0
50 ± 6
90
AH26 ~
:1
-
-
M e d i u m eluate
:2 :4 :8
-
<0.1 <0.1
0 7_+2 30+5
30 77 84
: 16 : 32
12.3 ± 1.30
< 0.1 0.3
68 ± 7 76 + 13
89 95
: 64 :128 :256 :512
2.9 _+ 0.10 1.4±0.02 1.1 ± 0 . 0 1 1.0 ± 0.07
0.9 1.0 1.0 1.0
90 _+ 19 119±6 -
106 113 -
+
46
0
AH26 ~
:80
-
-
D M S O eluate
: 160
-
-
: 320 : 640 :1280 :2560 :5120 : 10240 : 20480 : 40960
2.0 ± 0.24 1.4 _+ 0.07 1.1 ± 0.04
<0.1 < 0.1 <0.1 0.4 0.8 0.9
1.0 _+ 0.07
h0
:1
.1 ± 0.03
1.1
121 + 15
83
:2 :4 :8
.1 + 0 . 0 2 .0 _+ 0.06 .3 _+ 0.21
1.2 1.1 0.8
95 -t-7 92 _+ 1 87 ± 5
83 81 83
: 16 : 32 :64
.0 + 0.14 .0 ± 0.03 .0 ± 0.09
1.1 1.1 1.1
113 + 16 120 ± 5 101 ± 4
102 96 101
:128
.1 ± 0 . 5 3
0.9
: 40
1.0 ± 0.01
1.0
: 80 : 160 : 320 :640 : 1280 :2560 :5120
1.2 ± 0.17 1.1 _+ 0.08 1.2 ± 0.11 1.3 ± 0.12 1.4 ± 0.14 1.1 ± 0.08 1.2±09.15
0.9 0.9 0.9 0.9 0.8 0.9 0.9
:81920 Apexit ~ medium
eluate
Apexit a D M S O eluate
(+)
layers of the HeLa cells were exposed for 90 min to the materials to be tested. All concentrations were tested in triplicate; with each set of experiments usually repeated three times.
-
0
22
45 ± 3 74 _+ 2 1 0 2 + 10 115 _ 5 121 _+5 92 _+ 8 -
58 80 93 99 95 98 -
-
-
96± -
l
25 ± 2
16
50 ± 1 71 ± 1 93 ± 1 97 ± 1 102 ± 7 122 ± 7 126_+9
+
+
94
(+)
28 55 63 69 94 90 92
Thereafter, the cells were washed by two rinses with fresh, pre-warmed medium and allowed to recover for 2 h. This was followed by addition of BrdU in a final concentration of 20 ~ M for 60 min.
J. H eil et al. / Mutation Research 368 (1996) 181-194
188 Table 3 (continued)
umu-test
Substance
N2 m e d i u m eluate
Induction rate
G r o w t h factor
Test result
1:1 1:2 1:4 1:8 I : 16 1:32
-
<0.1
(+)
3.5+0.16 1.4 ___0.02 1.2 +- 0.02 1.0 + 0.04 1.0 + 0.02 1.0 +- 0.01 1.0 +- 0.03
1:64
1 : 128
N2 D M S O eluate
Herculite ~ m e d i u m eluate
:40 :80 : 160 : 320 : 640 : 1280 :2560 :5120
1.2 1.1 1.1 1.0 1.0 0.9
1:1 1:2 1:4 1:8 1:16 1 : 32 1 : 64 1 : 128
Herculite ~ D M S O eluate
Sono-CEM ® m e d i u m eluate
DIT
Dilution factor
: 40 : 80 : 160 : 320 :640 : 1280 : 2560 :5120 : 10240 1: 1 1:2 1:4 1:8 1:16 1:32 1 : 64 1:128
_+ 0.06 +- 0.04 +- 0.02 -+ 0.02 + 0.01 _+__0.03
0.1 0.7 0.8 1.0 h0 1.0 1.0 < 0.1 <0.1 0.7 1.0 0.9 1.0 1.1 1.2
1.1 +_ 0.03 1.0 ± 0.05 1.1 _+ 0.04 1.4 _+ 0.30 1.1_+0.02 1.0 +- 0.05 1.0 ± 0.06 1.0_+ 0.14
1.3 1.3 1.2 1.0 1.3 1.2 1.2 1.0
1.2 1.2 1.2 1.4 1.2 1.2 1.2 1.2
1.0 1.0 1.0 1.0 0.8 0.9 0.9 1.0
_+ 0.05 +- 0.03 ± 0.03 _+ 0.34 _+ 0.15 +- 0.18 _+ 0.08 _+ 0.12
-
-
1.0 1.0 1.1 1.1 1.1 1.1 1.0 1.1
+ 0.02 + 0.01 +- 0.11 +0.03 +0.03 ±0.10 + 0.06 +0.19
1.3 1.2 1.1 0.8 1.2 1.2 1.1 0.9
Subsequently, the c e l l s w e r e f i x e d with e t h a n o l / a c e t i c a c i d / w a t e r ( 9 0 : 5 " 5 ) for 30 min at room temperature. The alcohol was poured off and 4 N HCI was added to the fixed cells for 10 rain to denature the DNA. Excess acid was washed away by
% D N A synthesis 0
0 61 56 107 103 118 88
-+ 3 +- 19 -+ 3 + 7 +- 18 +- 26
Cell count
Test result
44 35 48 90 100 113 109 102
+
+ 42 _+ 3 61 _+2 68 + 1 63 +- 3 113±3 102 +- 3 94 _+ 4
60 78 92 98 94 95 103
90+-26
Ill
115 _+ 16
119
107 +- 4 118 +- 27 95 _+ 6 120_+ 11 112+-6 132 +- 23
126 99 105 111 116 92
118_+ 1 108 _+ 6 110+4 108 +- 8 132+--8 112+-4 119+-5 103 +- 15
86 80 80 79 91 86 87 84
1t7--+ 12 106 +_ 2 113+7 134 _+ 7 1 1 6 ± 16 94_+ 10 101 _+ 4 123 + 9
84 98 104 84 99 111 113 94
rinsing the microplate twice with tap water. Then a 1 : 1500 dilution of a monoclonal anti-BrdU antibody (Bio Cell Consulting, Reinach, Switzerland) was added to the cells for 30 rain. After washing the cells three times with tap water, a 1 "500 dilution of
J. Heil et al. / Mutation Research 368 (1996) 181-194
189
Table 3 (continued)
umu-test
Substance Dilution factor Sono-CEM ~' DMSO eluate
Ionoseal ~ medium eluate
Vitrebond ~' medium eluate
DIT Growth factor
: 40 :80 : 160 : 320 : 640 :1280 : 2560 :5120
0.9 _+ 0.01 1.4 ± 0.14 1.2 _+ 0.13 1.3 _+ 0.19 1.2 ± 0.11 1.4+0.18 1.3 ___0.08 1.2 _+ 0.15
0.8 0.8 0.9 0.9 0.8 0.8 0.8 0.9
:1 :2 :4 :8 : 16 : 32 : 64 : 128
0.9+0.11 0.9 -t- 0.08 0.9 + 0.05 0.9-t-0.10 1.0 + 0.08 1.0 _+ 0.02 1.0 + 0.02 0.9 ± 0.07
1.2 1.2 1.1 1.1 1.2 1.1 1.1 1.1
:1 :2
:4 :8 : 16 :32 :64 : 128 : 256 :512 Vitrebond ~ DMSO eluate
Induction rate
1:40
1 : 80 1 : 160 1 : 320 1 : 640 1:1280 1 : 2560 1:5120 1:10240 1 : 20480
-
-
-
-
Test result
%DNA synthesis
Cell count
-
-
18 + 8 43 _+ 6 84 +_ 2 96 _+ 2
5.9_+ 1.12 6.0 _+ 0.67 4.7 _+ 0.06 4.1 +0.12 3.3 _+ 0.12 2.3 + 0.08 2.0 _+ 0.03 1.6+0.10
0.2 0.4 0.6 0.8 0.9 0.9 0.9 0.9
6.2 _+ 0.21 4.5 _+ 0.24 3.1 _+ 0.23 2.0 _+ 0.23 1.7 _+ 0.22 1.4+0.10 1.2 _+ 0.04 1.1 -t- 0.04 1.1 + 0.04 1.1 _+ 0.07
0.5 0.6 0.7 0.9 1.0 0.9 1.0 1.1 1.0 1.0
53 68 78 78
-
+
Test result
-
103 _+ 15 96 _+ 7 89 _+ 6 88 + 3 121 _+ 11 111 _+ 13 119+8 109 + 3
84 83 83 80 97 97 93 90
0 0 3_+3 42 + 3 92 _+ 3 87 + 10 90_+8 99 _+ 16
59 76 94 85 101 115 119 95
123 _+ 10
90 97 97 88 111 104 99 84
110+2 109 _ 2 115+8 123 ___ 17 126 + 3 116_+ 10 98 _+ 12
peroxidase-conjugated F(ab)2-sheep-anti-mouse IgG
e l u t i o n o f the d y e w i t h T r i s b u f f e r a n d c o l o r i m e t r i c
a n t i b o d y ( S i g m a ) w a s a d d e d f o r a n o t h e r 30 m i n . T h e
measurement
cells were w a s h e d three times with tap water, and a
standard
freshly prepared peroxidase substrate solution was
o x i d e ) w a s u s e d as p o s i t i v e c o n t r o l .
added. The color development stop solution (H2804).
at 5 6 4
genotoxin
nm.
I n all e x p e r i m e n t s ,
4-NQO
(4-nitroquinoline
the N-
was stopped with a
T h e e x t i n c t i o n o f the w e l l s
w a s m e a s u r e d at 4 9 5 n m u s i n g a n E L I S A
reader.
2.4.2. Rating o f D I T test results The OPD/SRB
r a t i o s f o r d r u g - t r e a t e d c e l l s are
Cell c o u n t s w e r e d e t e r m i n e d b y s u l f o r h o d a m i n e B
expressed
( S R B ) a d s o r p t i o n to total cell p r o t e i n , f o l l o w e d b y
i n t o D N A . T h e r e s u l t s o f t h e t e s t s a r e g i v e n as m e a n
as p e r c e n t a g e s
of control
incorporation
190
J. Heil et al. / Mutation Research 368 (1996) 181-194
of triplicate determinations _+ standard deviation. If a Dis0 value (cell count corrected DNA synthesis of 50%, as compared to untreated controls) could be derived, the carcinogen-induced DNA synthesis inhibition is considered 'positive'. Samples showing no resp. moderate signs of concomitant cytotoxicity (cell count > 40%) are scored 'sufficient positive', the presence of higher degrees of cytotoxicity (cell count < 40%) sets scoring to 'limited positive'.
2.5. bl vivo-assay
recovered by shaking them in 5 ml of elution buffer for 2 h, using an Eppendorf shaker (1500 U/min). Aliquots of 0.2 ml were taken for the fluorometric
Table 4 A F E (in v i v o alkaline filter elution t e c h n i q u e ) g e n o t o x i c i t y results o f c h e m i c a l m o n o s u b s t a n c e s present in dental materials and extracts o f dental materials (for details, see Section 2: Materials and methods) Substance
2.5.1. Alkaline elution technique The alkaline elution technique assays for DNA strand breaks which are the most frequent consequence of exposure of cells or live animals to DNA damaging substances. Filter elution was performed according to the procedure of Kohn et al. (1976), which had been adapted to gills of freshwater clams Dreissena polymorpha and Corbicula fluminea by Waldmann et al. (1995). The use of clams or other lower invertebrates avoids experimental restrictions given by official directions. In alkaline elution experiments, clams (18-20 mm long, weighing 2.2 to 2.5 g) were exposed in 200 ml of 'low-salt water' supplemented with the extracts of dental materials. Circulation of water during exposure was provided by magnetic stirrers. Four specimens of clams per assay were incubated for 2 h in light-protected 500-ml glass beakers. After exposure, the gills of the clams were removed, immediately homogenized and the homogenate was transferred onto a filter apparatus for alkaline elution analysis. Aliquots from the homogenate were layered onto 0.22-/xm pore size polyvinyl fluoride filters (25 mm, Millipore), washed with 5 ml ice-cold PBS and then lysed by percolating 4 ml of a solution containing 2 M NaCI, 0.3% sodium lauroyl sarkosine, 0.02 M EDTA and 0.5 m g / m l proteinase-K (pH 8.7). The lysed homogenate residues on the filter were washed with 10 ml of a 0.02 M EDTA solution (pH 8.7). This procedure was followed by alkaline elution with tetraethylammoniumhydroxide buffer (pH 12.4) and 0.02 M EDTA. The flow rate of the elution was 0.04 ml/min; six 1.8 ml fractions were collected per channel. After elution, the DNA from the filters was
Concentration
A F E factor
[mM]
2-h incubation
24-h incubation
Test result
BHT Bis-GMA CQ CSA DCHA
2 2 2 2 2
1.00 1.07 1.15 1.10 1.25
(+ )
DMAPE DMBZ HEMA
2 2 2
0.92 1.27 1.0
(+ ) -
TPSb UDMA
6 2
O.7 1.24
+ (+ )
Dilution factor AH26 ~
1:40
1.84
-
( m e d i u m extract)
1:80 1 : 2500
1.45 -
1.32
A H 2 6 ~' ( D M S O extract)
1 : 60
1.31
Apexit ~ ( m e d i u m extract)
1 : 27 1 : 118
1.05 0.84
N2
1 : 143
0.88
Herculite ~ ( m e d i u m extract)
l:27
1.02
Sono-CEM * ( m e d i u m extract)
1:27
0.96
Vitrebond e ( m e d i u m extract)
1 : 27 1:50 1 : 67
1.67 1.38 1.06
-
1 : 1000
-
1 : 2500
-
1.33 1.09
1:1000
-
1.24
( m e d i u m extract)
Vitrebond ~ ( D M S O extract)
+
-
-
191
J. Hell et al, /Mutation Research 368 (1996) 181-194
D N A determinations of the D N A eluted in the fractions. Fluorescence was measured by an automated version of the procedure described by Stout and Becker (1982).
3. Results Tables 2 - 4 survey the results of the genotoxicity testing of 14 chemical monosubstances present in dental materials and 7 extracts. It's striking that most agents tested yield 'positive' results in at least one of the genotoxicity tests; however, with effects ranging from 'borderline' to 'strong positive'. The higher percentage of 'positives' in the eucaryotic DIT, to a certain degree, can be attributed to a concomitant high toxicity of many compounds which kills most tester bacteria and counteracts positive outcomes in bacterial genotoxicity/mutagenicity tests. Umu growth factor values > '1' indicate that such sub-
2.5.2. Rating of afe test results The slope factors of the D N A alkaline elution curves, indicating the number of single-strand breaks in the D N A from the treated animals, in relation to their appropriate controls, are expressed as arbitrary units ('AFE-factor'). A factor > 1.3 is used as an indicator for a 'sufficient positive' genotoxic potential of a substance ('limited positive' = factor > 1.2). Slope factors < 0.7 are indicative of D N A - D N A crosslinks and scored as 'sufficient positive'.
--=--
f
induction rate
....... Vo,,~ h M d o r
- - = - - induction rate ......... grow~l ~ ' o r
5
• 1,2 14-
- 1,4
T 12-
AH26
10-
......
medium
~
e~tmct
/
umu
-1.o
4-
- 1,2 umu
-0,8
- 0,6
g -
~-~
-1.0
Herculite medium ¢dract
,0.6 ~
- 0,4 ~
• 4-
.0,4 1-
-0,2
20 0,001
i
. . . . . . . .
. . . . . . . .
• 0.2
0,0
0,01
. . . . . . . .
0 • ' '0.01
0,1
. . . . . . .
i 1
01,1
dilution factor
dilution factor
- - = - - D N A syndlesi$ cell count
1
--I--
......
J
~2o~
140-
100 i
~2o-"
T •
........
~
.....
DNA s~thesis cell count
T DIT
.
.m 100 i
"~ 80 O~ 60-
'3£\
7< 4oO A1-126
2o~
medium ~dmet
01 ....
i
0,01
\,
. . . . . . . .
i
0,1
dilution factor
r i O.O
. . . . . . . .
.~,
80-
Z a
60-
•
4o-
-
20-"
i
1
O"
Herculite m e d i u m eoctm~
........
, ,,,i
;
dilution factor
Fig. 1. Effects of the medium extracts of AH26 ~ and Herculite '~ in u m u - t e s t and D N A synthesis inhibition test (DIT).
1
I
J
192
J. Heil et al. /Mutation Research 368 (1996) 181-194
I
--m-- inductionrate1 growth~¢torI
--=-- DNAsyn~esis cell count
. . . . . . . .
1.2 120-
1,oo
DIT
100'~ 00.
1 0,4 0,6
~
60-
z
4o.
\,
2-
Vitrebond me01umextract
0,2 0 0.001
.
.
.
.
.
.
.
0.~1
.......
0'.1
•
0.0
dilution factor
....
i
o,01
. . . . . . . .
i
o,1 dilution factor
Fig. 2. Effect of the medium extract of Vitrebond ~ in umu-test and DNA synthesis inhibition test (DIT).
stances (Bis-EMA, Bis-GMA, UDMA) are bonding agents which make scoring more difficult. In the case of the widely used dental materials AH26 ® and Vitrebond ®, the in vivo - tests with the alkaline elution method confirmed the in vivo - relevance of 'strong positive' in vitro results.
4. Discussion Genotoxlcity is one of the many types of adverse effects of chemicals. Since most dental materials release small amounts of various substances into their physiological environment (pulp, oral cavity), appropiate regulations have to ensure that the concern about the (possible) genotoxicity/mutagenicity/carcinogenicity of dental materials is abolished or at least minimized. The problem connected herewith is less the analytical determination of a substance but more "what is the concentration of the substance that causes harm?". The recent European Standard "Biological evaluation of medical devices, Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicity" (EN 30993-3, ISO 10993-3:1992) recommends methods which are listed in the "OECD Guidelines For The Testing Of Chemicals". For certain aspects, alternative methods can be accepted. Nevertheless, screening tests should be performed 'state of the art', i.e., tests using microplates should be preferred, allowing for the fast, easy and simultaneous testing of extensive numbers of data points. Since many dental
materials are highly toxic (Leinenbach et al., 1993; Karbakhsch et al., 1994), it is a basic requirement for genotoxicity tests to easily quantify simultaneous cytotoxicity in order to avoid misinterpretation of the data. The umu-test and the DNA synthesis inhibition test both fulfil this criterion, whereas, e.g., Ames test results are usually published without adequate toxicity data. Moreover, it cannot be excluded that socalled 'small colonies' (i.e., survivors of the toxic effect of the test material which grow by using up the histidine in the top agar) are counted as revertants (Stea et al., 1994). The root canal filling material AH26 ® has already been reported to be mutagenic in the Ames test. These findings could be confirmed by strong genotoxic responses in the bacterial umu-test (Fig. 1), the eucaryotic DIT (Fig. 1) and the in vivo induction of DNA fragmentation, as measured by the alkaline filter elution technique (Table 4), indicating that the DNA-damaging properties of AH26 ® have to be taken serious. It is remarkable that in the umu-test AH26 ® features genotoxic responses up to a 10000-fold dilution when extracted with DMSO, whereas the medium extract is less genotoxic by a factor of about 100. These findings are equivalent to Stea et al. (1994) and Schweikl et al. (1994) who report the mutagenicity of DMSO extracts of AH26 ® to Salmonella typhimurium. In contrast to Schweikl et al. (1994) but in accordance with Stea et al. (1994) is the observation that mutagenic substances of AH26 ® (presumably an epoxy-derivative of bisphenol-(A)-diglycidylether) are present even in the poly-
z Heil et al./Mutation Research 368 (1996) 181-194
merized material. This supports the hypothesis that mutagenic agents may migrate into the surrounding tissue. Although human exposure will be maximal in the first hours after implementation, a prolonged exposition time may drastically reduce the concentrations required for significant genotoxic effects, as shown in the AFE (Table 4). Vitrebond ® is a glass ionomer cement which elicits strong genotoxic responses in all three test systems (Fig. 2, Table 4). In the u m u - t e s t , the least significant dilution of the extract is about 1000fold in both the DMSO and medium extract. In the DIT, however, only the extract with physiological cell culture medium caused effects indicative of genotoxic activity, whereas the DMSO extract didn't lead to a significant inhibition of DNA synthesis, presumably because of it's higher dilution due to the toxic effect of DMSO itself. In contrast to AH26 ®, the genotoxic effects of Vitrebond ® are nearly unaffected by accompanying cytotoxic effects. In the AFE, a prolonged exposition time (24 h vs. 2 h) reduced the concentrations required for significant genotoxic effects by a factor of approx. 20 (Table 4). Zinc-Eugenol cements like N2 ® are used in general dental practice as a filling of root canals. Eugenol (4-allyl-2-methoxyphenol, a natural substance from the oil of cloves) was previously found to behave as a genotoxin in several in vitro systems including the Ames test (RTECS, 1995). In DIT and u m u - t e s t , extracts of N2 ® produced effects indicative of genotoxic activity, too. Whether Eugenol or another substance is the causative agent for this, remains to be established. CQ is a substance which is sufficient positive in both the u m u - t e s t and the DIT. There is no concomitant cytotoxicity which could interfere with the genotoxicity measurements and even the effective concentrations are in the same range. CSA, a derivative of CQ, is sufficient positive in the DIT, but since the u m u induction rates > 1.5 are accompanied with growth factors < 0.5, the u m u effects have to be taken with caution. TPSb is negative in the u m u - t e s t . Higher concentrations ( > 10 mM) lead to a complete inhibition of procaryotic as well as eucaryotic cell metabolism. In the DIT, TPSb shows a 'classic' concentration-inhibition curve, ranging from 5 mM to 0.04 mM. Hence, TPSb has to be classified as sufficient posi-
193
tive in the DNA synthesis inhibition test. Furthermore, the result of the in vivo - alkaline elution assay supports the hypothesis that TPSb may possess a genotoxic potential, presumably by causing (proteinase-K resistant) DNA-DNA crosslinks. Herculite ® is an example for a dental composite which gave negative results in both DIT and u m u - t e s t (Fig. 1). Interestingly, Herculite ®, according to manufacturer's description a composition of 16 components, contains substances which have been tested 'positive' in DIT a n d / o r u m u - t e s t (Bis-EMA, BisGMA, CQ, CSA, DEGDMA, TPSb). The reason for this discrepancy in the outcome of the tests certainly is complex ('modulation'), but it cannot be excluded that simple concentration effects contribute to this phenomenon. HEMA, which has wide applicability in medical technology, was not genotoxic under the conditions of the three bioassays used. The apparent non-genotoxicity of some substances, however, may be due to insufficient metabolic activation by the tester organisms. Nevertheless, additional tests with a liver microsomal activation system ($9 mix) were not performed, since the dental materials investigated in this study do not contain molecular structures which could be activated by $9 mix. Furthermore, pre-studies have given evidence that the presence of $9 reduces effects (data not shown). Non-genotoxic carcinogens, many of them presenting strong cytotoxic properties, induce tumors via an indirect threshold mechanism. Since the overstimulation of cell division (mitogenesis) enhances tumor production (Ames and Gold, 1990), the continued irritation of the oral tissues adjacent to certain dental materials may be seen as an additional risk factor. Of course, this does not automatically involve serious health consequences, but reducible hazards should be eliminated. The point in question normally involved with in vitro-tests is their extrapolation to any human risk connected therewith. Many mathematical models have been advanced to carry out the extrapolation from in vitro results to small doses in vivo, but most assumptions on the hazards from small amounts of carcinogens are unverifiable. Taken together, the results of this study give support to considerations that besides their benefits several dental materials possess unwanted properties
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that can be a threat to human health. Fortunately, there are safer alternatives available, particularly when one bears in mind that many composites are not only interchangeable, but made out of the same set of monosubstances. In the long run, final biological evaluation of dental materials has to be done by a group of experts on the basis of published results and adequate knowledge.
Acknowledgements This stuty was supported by a grant of the DFG (Ge 455/4-2).
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