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Mutagenicity of ethanolic extracts of used acrylic dentures
Genetic Toxicology
ELSEVIER
Mutation Research 321 (1994) 241-251
Mutagenicity of ethanolic extracts of used acrylic dentures D.M. Parisis a, N.L. Eskoz b, W.G. H e n d e r s o n c,. Department of Periodontics, Biochemistry and Microbiology, Loyola Unicersity, School of Dentistry, Maywood, IL, USA t~Dental Sercice, and c V.A. Cooperatil,e Studies Program, Hines V.A. Hospital, Hines, IL 60141-5151, USA (Received 14 April 1993; revision received 4 January 1994; accepted 6 January 1994)
Abstract The in vivo physicochemical sorption of mutagenic substances onto acrylic polymers was investigated in worn acrylic dentures. Thus, ethanolic extracts of acrylic dentures from 41 of a total of 69 human donors (60%), were found mutagenic in the standard plate incorporation Salmonella mutagenicity test against either TA98 or TA100 strains. Denture extracts from smokers produced mutagenicity more often than the ones from non-smokers (75% vs. 45%, P 0.01). Mutagenicity was preferentially directed against TA98 (TA98:TA100 = 2.9 : 1, P < 0.0005). Predilection for TA98 was more pronounced in denture extracts from non-smokers (4.7 : 1) than from smokers (2.0 : 1). When direct mutagenicity was observed, it was reduced by the rat-liver $9. Induced mutant yields were 6.1 + 3.9 and 7.0 _+ 8.9 times higher than the spontaneous for TA98 and TA100 respectively (smokers, 50-cm 2 denture surface area e q . / p l a t e + $9). Denture extracts from smokers induced higher levels of mutation than the ones from non-smokers (TA98 + $9, smoker:non-smoker = 2:1, P < 0.01). Mutagenicity was associated with longer periods of denture usage ( P 0.007). Thus, denture poly(methyl methacrylate) base material can adsorb mutagenic substances, possibly from diet and tobacco, which are extractable by ethanol. Theoretically, the in situ alcoholic desorption and recirculation of carcinogenic mutagens may have a contributory role in certain cases of intra-oral and upper alimentary tract carcinogenesis.
Key words: Acrylic dentures; Mutagen adsorption; Oro-pharyngeal carcinogenesis
1. Introduction
* Corresponding author: Dr. W.G. Henderson, c / o Dr. Dimitrios Parisis. This work was supported by the School of Dentistry, Loyola University of Chicago and Hines V.A. Hospital, Hines, IL, USA. AbbretJiations: HPLC, high-performance liquid chromatography; DMSO, (di)methyl sulfoxide; NPD, 4-nitro-l,2-phenylenediamine; B[o~]P, benzo[a]pyrene; PMMA, poly(methyl methacrylate). Elsevier Science B.V. SSDI 01 65-1 2 1 8 ( 9 4 ) 0 0 0 0 3 - L
A n i n t e g r a l p a r t o f the analysis of o r g a n i c m u t a g e n s in c o m p l e x m i x t u r e s is t h e i r e x t r a c t i o n a n d c o n c e n t r a t i o n (Claxton et al., 1992; S c h u e t z l e a n d Lewtas, 1986). T h e p r i n c i p l e o f s o r p t i o n o n t o p o r o u s o r g a n i c p o l y m e r s has b e e n successfully e x p l o i t e d ( D r e s s l e r , 1979) Thus, in c o m b i n a t i o n with m u t a g e n i c i t y assays, m u t a g e n analysis by s o r p t i o n o n t o A m b e r l i t e p o l y m e r i c a d s o r b e n t se-
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D.M. Parisis et al. / Mutation Res'earch 321 (1994) 241 251
ries (e.g. X A D macroreticular acrylic resins) proved very fruitful in the detection and isolation of many organic mutagens in a variety of matrices (Grabow et al., 1981; Claxton et aI., 1992; Vennit, 1988). Acrylic denture base material is also a resin, a poly(methyl methacrylate). Denture resin has no macroreticular structure (Phillips, 1982), but otherwise it is chemically identical to XAD-8 Amberlite resin (Dressier, 1979) which is used for sorption-based analytical p r o c e d u r e s m e n t i o n e d above. It follows that orally transient mutagenic substances from tobacco smoke and diet for example, may become adsorbed onto the denture surface, resulting in retardation of their passage through the oral cavity. Their subsequent in situ desorption by alcohol may produce changed exposure patterns which might be a contributing factor in certain cases of oral and upper alimentary tract carcinogenesis (Stich et al., 1983; Khojasteh and Kraybill, 1988). The purpose of this study was to examine whether worn dentures from human subjects contain adsorbed mutagens that could be desorbed through the action of alcohol, by assessing the mutagenic activity of their ethanolic extracts in the S a l m o n e l l a / m i c r o s o m e mutagenicity test.
2. Materials and methods Chemicals
Ethyl alcohol-U.S.P. Dehydrated 200 Proof Punctilious was obtained from Q u a n t u m Chemical Corp., U.S.I. Division, Tuscola, IL. Methanol, m e t h y l e n e chloride and hexane, all high p u r i t y / H P L C grade, were from Burdick and Jackson Division of Baxter Healthcare Corp., Muskegon, MI. DMSO, H P L C grade and the diagnostic mutagens 4-nitro-l,2-phenylenediamine (NPD) and benzo[c~]pyrene (B[c~]P) were obtained from Aldrich Chemical Co., Milwaukee, WI. Agar was from Difco Laboratories, Detroit, M1 and Oxoid Nutrient Broth No. 2 from Oxoid LTD., Basingstoke, Hants., England. O t h e r chemicals and biochemicals were purchased from Sigma Chemical Co., St. Louis, MO, Mallinckrodt Inc., Paris, KY and J.T. Baker Chemical Co., Phillipsburg, NJ.
Table 1 Relevant data used from the history of denture donors Denture age (years worn) Denture-cleaning habits (cleansing solutions) Denture repair (relining) Tobacco-smokingstatus Alcohol consumption Coffee and/or tea consumption
Denture collection
Complete upper a n d / o r lower dentures were obtained from dental patients (donors) at Hines V.A. Hospital Dental Service Clinic. New dentures were made in exchange for the worn ones that were relinquished for use in this study. A brief patient history was taken with particular emphasis on items listed in Table 1. Collected dentures were wrapped in aluminum foil and stored at - 2 0 ° C until processed. Dentures from a total of 69 donors are included in this study: upper and lower dentures from 36 donors, upper denture from 31 donors and lower denture from 2 donors. Denture mutagen extraction
All procedures were performed in dim light to prevent breakdown of any photosensitive mutagens. Whenever donors contributed 2 denture pieces, both upper and lower dentures were pooled, processed and tested together. At no time were dentures from different donors ever mixed. First, the total surface area of each denture (both tissue fitting and non-fitting surfaces) was determined by covering with aluminum foil, tracing on standardized graph sheet (Keuffel and Esser Co., Morristown, N J) and weighing the cut-outs. Mean total surface area of pooled upper and lower dentures (n = 36), upper denture (n = 31) and lower denture (n = 2) was 174 _+ 24, 102 _+ 15 and 63 cm 2 respectively. Dentures were rinsed off with deionized water to free them from any gross debris. The denture(s) from each donor were then extracted with absolute ethanol, at 1 ml e t h a n o l / c m e denture surface area, in one-liter stoppered glass jars, by shaking in an incubator ( S / P Ultra-Tech Gold Series Shaking Water Bath BT-47, Baxter Healthcare Corp., McGaw Park, IL) at 37°C and 125 s t r o k e s / m i n for 1 h. This
D.M. Parisis et al. / Mutation Research 321 (1994) 241-251
extraction was identically repeated 2 more times with fresh ethanol and the 3 successive extracts were pooled for each donor. The pooled ethanol extracts were passed through a 0.22 /~m MF-Millipore GS type filter (Millipore Sterifil Aseptic System, 47 mm) and then evaporated to dryness in a rotary evaporator (Yamato RE-51, Baxter) at 45°C. The residue was then redissolved in 1-1.5 ml of methanol which was then added to 30 ml of sodium phosphate buffer, 20 Mm, p H 7.0. This buffered mixture was then twice extracted with 2 volumes of hexane in a 250-mi separatory funnel using a Wrist Action Shaker, model 75 (Burrell Scientific Co., Pittsburgh, PA) at approximately 420 oscillations/min for 5 min. each cycle. These hexane extracts were discarded. The hexane preextraction step proved to limit the bacteriocidal effects of the extracts (hexane removes bacteriocidai and antimutagenic fatty acids with minimal extraction of pyrolysis mutagens) (Stich et al., 1982; Hayatsu et al., 1983; T a k e d a et al., 1991). The buffered mixture was subsequently extracted 3 times with 2 volumes of methylene chloride (Nielsen, 1992), under similar conditions. These extracts were pooled and saved. Then, the p H of the aqueous mixture was adjusted with N a O H to 10.0 and the methylene chloride extraction procedure was again applied (2 extractions). Finally, the p H of the mixture was readjusted with HC1 to 4.0 and the methylene chloride extraction procedure was repeated once again. Pooled methylene chloride extracts (for each donor) were combined and dried over N a z S O 4. The combined extracts were then rotary evaporated to dryness. The final residue was then redissolved in D M S O and the volume was adjusted to 1 /zl D M S O / c m 2 denture surface area. The residue was then assayed for mutagenicity in the A m e s test. The appearance and the net weight of the residues were routinely recorded.
Bacterial tester strains Salmonella typhimurium
strains TA98 and TA100 were kindly supplied by Dr. Bruce N. Ames of the University of California at Berkeley. These tester strains were grown, tested and maintained according to the revised methods of Maron and Ames (Maron and Ames, 1983).
243
$9 fraction preparation Rat-liver $9 fraction was prepared based on the procedure of G a r n e r et al. (1972). Young male S p r a g u e - D a w l e y rats (Harlan S p r a g u e Dawley, Indianapolis, IN) weighing approximately 200 g each were used after induction with Aroclor 1254 (The Foxboro Co., Ultra Scientific, North Kingston, RI). The protein concentration of this $9 fraction was adjusted to 28 m g / m l and it was stored at - 8 0 ° C until used.
Mutagenicity assay The standard plate incorporation protocol of the Salmonella mutagenicity test was used without modification throughout this study (Maron and Ames, 1983). All procedures were also carried out in dim light. Briefly, denture extract residues corresponding to 25- and 50-cm 2 denture surface area in a total of 0.1 ml of D M S O were mixed with 0.1 ml of a standardized late exponential culture of tester strains grown the previous night (1-2 x 108 cells/plate) and 2 ml of top agar in the presence and in the absence of 0.5 ml of $9 mix (or sodium phosphate buffer, 0.1 M, pH 7.4) and poured onto minimal glucose plates. Due to the limited size of the individual dentures, the magnitude of mutagenic activity, testing against 2 strains sometimes in the absence of $9, the designed test concentrations of the extracts were not tested more than twice to produce S.D. The plates were incubated for 48 h in a 37°C dark incubator and then scored for revertant colonies. N P D ( 2 0 / z g / p l a t e ) and B[a]P (1 /xg/plate) were used in every assay (triplicates) as diagnostic mutagens. Extract concentrations with toxic (thinning o f / o r no background lawn) scores were re-. jected. No increase in the density of the bacterial lawn was ever observed to indicate interference by any histidine or its precursors in the final residue sample. A positive mutagenic response was considered when there was a doubling in the mutant yield (number of r e v e r t a n t s / p l a t e ) over the control without the extract residue.
Statistical analysis In comparing tester strains within the same individual dentures, M c N e m a r ' s chi-square test for correlated proportions was used. In making
D.M. Parisis et al. / Mutation Research 321 (1994) 241-251
244
comparisons of mutagenicity between smokers and non-smokers and other variables (denture cleaning habits, denture repair, alcohol, coffee and tea beverage consumption) the chi-square test for independent samples was used. In comparing mutant yield ratios between concentrations within each tester strain, the paired t-test was used. To compare mutant yield ratios between smokers and non-smokers and for the association of mutagenicity and degree of denture usage the t-test for independent samples was used. The relationship between pack-years of tobacco usage and mutant yield ratios was examined using Pearson's product-moment correlation coefficient. This relationship was also examined partialing out the effect of years of denture usage using the partial correlation coefficient.
3. Results
Table 2 Historic ~' spontaneous and induced mutant yields Tester
N umbe r of revertant c o l o n i e s / p l a t e
strain
-$9
+$9
TA100
146+23
142_+17
666+ 88
974+_22//
25+_ 6
39+_13
1680+162
254+_ 68
TA98
NPD h
Blot]P+ $9 c
" n = 12 assays. b 21) # g / p l a t e . " 1 ~zg/plate.
in the presence of $9 from 63 donors at extract concentrations corresponding to 50-cm 2 denture area e q . / p l a t e , except for the open symbols which correspond to 25-cm 2 eq./plate. 18 denture extracts within the non-smoker subgroup and 15 extracts within the smoker subgroup were tested against both strains (4 extracts from donors of questionable smoking status and 2 other extracts not tested with $9 are not shown). MYR of indi-
Results from dentures from a total of 69 donors are presented in this study. Mutagenicity is reported as the mutant yield ratio (MYR) of the experimental over the control, according to the formula:
28-27-/
1817-
D-( S9-sp ) MYR -
16-
sp
15I
where D is the mutant yield of the plates containing the denture extract residue, $9 the mutant yield of plates containing $9 mix plus D M S O and no extract and ap the spontaneous mutant yield of plates containing DMSO, buffer and neither $9 nor extract. MYR > 2 was considered a positive mutagenic response. Table 2 shows the historic spontaneous and NPD- and Bier]P-induced mutant yields of controis concurrently tested with the denture extracts. Because there was a significant association between mutagenicity and smoking status (denture extracts from 75% of smokers had MYR > 2 vs. 45% from non-smokers, P 0.01), results are shown separately for the smoker and non-smoker denture donor subgroups. Fig. 1 summarizes the results. It shows the mutant yield ratios produced by denture extracts
o 14--
rl- 1 3 - 12-11-10--
:E
9
--
8
--
7
--
6
--
5
--
4
!
•
I
I
I-
-i
3
I
--
2
TA100
TA98
Non-Smokers
TA100
TA98
Smokers
Fig. 1. Mutant yield ratios (extract:control) produced by denture extracts, in the presence of $9, from 63 donors. Extract concentrations correspond to 50-cm 2 denture area e q . / p l a t e except for the open symbols which correspond to 25-cm 2 e q . / p l a t e . For each strain only one concentration is shown.
D.M. Parisis et al. / M u t a t i o n Research 321 (1994) 241-251 Table 3 Percentage of donors with mutagenic denture extracts a Group (n = number of donors)
TA100
TA98
TA100 and/or TA98
23.1 (52)
76.5 (51)
59.4 (69)
Non-smoker
10.3 (29)
68.2 (22)
45.4 (33)
Smoker
42.1 (19)
78.6 (28)
75.0 (32)
29.4 (34)
85.3 (34)
-
Non-smoker
16.7 (18)
77.8 (18)
-
Smoker
46.7
93.3
-
(15)
(15)
GROUP I b All
G R O U P II c All
Tested with $9 (2 extracts tested against TA98 without $9) at concentrations corresponding to 25- a n d / o r 50-cm 2 denture surface area eq./plate. Mutagenic response considered positive when mutant yield ratio of experimental over control was greater than 2. h All the donors; individual extracts not necessarily tested against both strains; it also includes 4 donors of undetermined smoking status. TA100, non-smoker vs. smoker P 0.01; TA98, non-smoker vs. smoker P 0.4; TA100/TA98, non-smoker vs. smoker P 0.01. c Select donors from G R O U P I whose extracts were concurrently tested against both strains. All, TA100 vs. TA98 P < 0.0005; TA100, non-smoker vs. smoker P 0.05; TA98, nonsmoker vs. smoker P 0.18; non-smoker, TAI00 vs. TA98 P < 0.005; smoker, TA100 vs. TA98 P < 0.01.
vidual denture extracts ranged from 2.1 to 17.6 and 2.1 to 27.2 for TA98 and TA100 respectively. Overall, induced mutant yields reached a 6.1 _+ 3.9-fold and 7.0 + 8.9-fold increase over the spontaneous with TA98 and TA100 respectively (smokers, at extract concentration corresponding to 50-cm 2 denture surface area e q . / p l a t e ) . Table 3 shows the percentage of denture donors with mutagenic denture extracts against TA100 a n d / o r TA98, at 25- a n d / o r 50-cm 2 denture surface area e q . / p l a t e , in the presence of $9 (2 extracts were tested against TA98 in the absence of $9). G R O U P I represents all samples. Individual denture extracts were not necessarily
245
tested against both tester strains and it includes also 4 donors of undetermined smoking status. G R O U P II contains select donors from G R O U P I whose denture extracts were concurrently tested against both strains. Thus, denture extracts from 59.4% of the donors were found positive for mutagenicity. Results with 34 denture extracts that were concurrently tested against both strains ( G R O U P II) showed that extracts were more likely ( P < 0.0005) to be mutagenic against TA98 (85.3%) than against TA100 (29.4%) (TA98: TA100 ratio of 2.9:1). G R O U P I also showed a similar qualitative trend with 76.5% of them mutagenic against TA98 and 23.1% against TA100 (3.3 : 1). Within G R O U P I, denture extracts from smokers were more likely ( P 0.01) to be mutagenic on either strain (75.0%) than extracts from non-smokers (45.4%). This trend was particularly true ( P 0.01) with TA100 tester strain which showed a 4.1:1 smoker (42.1%) to non-smoker (10.3%) ratio of positive mutagenic response. The respective responses for smokers (78.6%) and non-smokers (68.2%) with TA98 showed no statistically significant difference between them ( P 0.4). Within each subgroup, results from denture extracts tested against both strains ( G R O U P II) showed a predilection for mutagenicity against TA98 a trend already observed with G R O U P I. Thus, extracts from 77.8% of non-smoker donors were mutagenic against TA98 as opposed to 16.7% of them that were mutagenic against TA100 ( P < 0.005). For the smoker subgroup the respective percentages were 93.3% for TA98 and 46.7% for TA100 ( P < 0.01). It is interesting to note that the intra-strain differences in the response between the 2 subgroups were greater with TA100 ( P 0.05 and a smoker to non-smoker ratio of 2.8: 1), than with TA98 ( P 0.18 and a respective ratio of 1.2: 1), reflecting a similar trend within G R O U P I. Table 4 shows the mutagenic potency of the denture extracts that were tested in the presence of $9 against TA100 a n d / o r TA98, at 2 5 - a n d / o r 50-cm 2 denture surface area e q . / p l a t e . G R O U P I represents all the mutagenic extracts from either non-smokers or smokers. Individual extracts were not necessarily tested against both strains nor at both concentrations. Thus, at the 50-cm 2
D.M. Par±sis et al. / M u t a t i o n Research 321 (1994) 241-251
246
Table 4 M u t a g e n i c i t y ~' o f d e n t u r e e x t r a c t s at 25- a n d 5 0 - c m 2 denture area eq./plate p l u s $9 Group (n = n u m b e r o f donors) GROUPI
TA100 25 c m 2
TA98 50 cm 2
25 c m 2
Non-smoker
4.9±4.4 (3)
2.2 (1)
3.0±1.3 (7)
3.1+0.6 (14)
Smoker
4.9±4.9 (5)
7.0±8.9 (7)
4.2±2.3 (14)
6.1+3.9 (17)
Smoker
Smoker
-
-
2.5 + 0 . 2
3.5±0.5
(6)
(6)
5.5+5.4
10.4+11.2
4.6± 2.4
7.1+4.3
(4)
(4)
(11)
(11)
5.5±5.4 (4)
7.4±9.7 (6)
5.9+3.6 (4)
7.4±5.2 (6)
III d
'~ Mutagenicity expressed as the mutant yield ratio of experimental ( d e n t u r e e x t r a c t ) o v e r c o n t r o l ( D M S O + $ 9 ) + SD. b All the mutagenic extracts (with $9, f r o m either non-smokers o r s m o k e r s ) ; individual dentures not necessarily tested against b o t h strains nor at b o t h c o n c e n t r a t i o n s . T A 9 8 at 25 c m 2, n o n - s m o k e r vs. s m o k e r P > 0.2; T A 9 8 at 50 c m 2, n o n - s m o k e r vs. s m o k e r P < 0.01. ~ M u t a g e n i c e x t r a c t s t e s t e d at b o t h concentrations against a particular strain. T A 9 8 n o n - s m o k e r o r s m o k e r , 25 c m 2 vs. 50 cm 2 P <0.01;TA100smoker, 2 5 c m 2 vs. 5 0 c m 2 P > 0 . 1 . d M u t a g e n i c e x t r a c t s c o n c u r r e n t l y tested against both strains at the same c o n c e n t r a t i o n . A t 25 c m 2 o r 50 c m z, T A 1 0 0 vs. T A 9 8 P > 0.8.
eq. concentration and with TA98, potency was higher (P < 0.01) in the smoker subgroup (1.96 : 1 smoker to non-smoker ratio). There are no adequate data with the non-smoker subgroup tested against TA100 at this concentration for a similar comparison. G R O U P II represents the mutagenic extracts that were tested at both concentrations against a particular strain. There was a dose-response relationship of potency in each subgroup with TA98. Thus, the 50-cm 2 eq.:25-cm 2 eq. dose ratio of MYR was 1.4:1 for the nonsmoker subgroup ( P < 0.01) and 1.5:1 for the smoker subgroup (P < 0.01). G R O U P III represents the mutagenic extracts (from smokers) that were concurrently tested against both strains at the same concentration. Thus, when MYR for the
E x t r a c t No.
- $9
+ $9
TAI00
l
3.3
2.2
TA98
2 3 4 5 6 7 8 9 10
2.3 2.5 3.0 < 2.0 < 2.0 < 2.0 2.7 9.2 15.8
2.1 2.3 7.0 2.5 7.0 11).5
c
Non-smoker
GROUP
Mutagenicity ~' o f select b d e n t u r e e x t r a c t s t h a t were tested in the presence a n d / o r in the absence of the $9 fraction Tester strain
50 c m 2
b
GROUPII
Table 5
" Mutagenicity as in T a b l e 4 ± $9. i, E x t r a c t s t h a t when tested with a n d / o r w i t h o u t $9 w e r e either directly a n d / o r indirectly m u t a g e n i c ; all f r o m smokers (except No. 8); all t e s t e d at 2 5 - c m 2 denture surface area eq./plate ( e x c e p t No. 7 a n d No. 8, t e s t e d at the 5 0 - c m 2 eq. concentration).
same concentration were compared no differences were found between the 2 tester strains (P > 0.8 for each concentration). In Table 5 shows that certain extracts when appropriately tested exhibited direct mutagenic activity or S9-mediated mutagenicity. All 4 extracts that were both directly and indirectly mutagenic yielded decreased mutagenic activity in the presence of the $9 metabolic system. Table 6 shows that positive mutagenic response was statistically correlated with the degree of denture usage (P 0.007). There was no significant relationship between pack-years and magnitude of mutagenic response even after partialing out the effect of denture usage. For TA98, the correlation coefficient of pack-years and mutagenic response at 25 cm: was r - 0 . 1 7 (n = 16, P 0.58) and at 50 cm 2 was
Table 6 R e l a t i o n o f mutagenic activity a of denture e x t r a c t s to the n u m b e r o f y e a r s o f denture usage
Mutagenic activity
Years + S D
(n = n u m b e r o f donors)
Negative (28) Positive (41) " Positive w h e n MYR > 2. Negative vs. positive P 0.007.
8 . 2 + 6.2 14.3 ± 11.5
D.M. Parisis et a l . / Mutation Research 321 (1994) 241-251
r - 0 . 0 0 3 (n = 18, P 0.99). After partialing out the effect of years worn, the partial correlation coefficient was r 0.09 ( P 0.81) and r 0.22 ( P 0.57) for 25 and 50 cm 2 respectively. For TA100 the corresponding coefficients were r - 0 . 1 3 (n = 7 , P 0.78) at 25 cm 2 and r - 0 . 0 6 ( n = 7 , P 0.91) at 50 cm 2. After partialing out, the partial coefficients were r 0.41 ( P 0.5) and r 0.43 ( P 0.47) respectively. No statistically significant correlation was found between mutagenicity and denture cleaning habits ( P 0.474), denture repair ( P 0.135), alcoholic beverage consumption ( P 0.845) and coffee or tea drinking ( P 0.85).
4. Discussion
This study reveals that a significant percentage of our denture donors (60%) wore dentures whose methylene chloride-fractionated ethanolic extracts contained substances mutagenic towards the Salmonella tester strains TA98 and TA100. Denture extracts from smokers were more likely to be found mutagenic, against either strain, than the ones from non-smokers (75% vs. 45%, P 0.01); however, when frequency of occurrence was measured separately for each strain, this relationship attained statistical significance with TA100 strain only (smoker 42% vs. non-smoker 10%, P 0.01). Mutagenicity was preferentially manifested with the frame-shift mutation detector strain TA98 than with the base-pair mutation detector strain TA100 (TA98:TA100 = 2.9 : 1, P < 0.0005). This predilection for TA98 was more pronounced in the non-smoker subgroup (4.7:1, P < 0.005) than in the smoker subgroup (2.0 : 1, P < 0.01). Mutagenic potency was 2 times higher in smokers than in non-smokers (TA98, at 50-cm 2 denture surface area equivalent per plate and in the presence of metabolic activation, P < 0.01). Overall, induced mutant yields were 6.1 _+ 3.9 and 7.0_+ 8.9 times over the spontaneous for TA98 and TA100 respectively (smoker denture donors, at 50-cm 2 denture surface area e q . / p l a t e + $9) Both direct and rat-liver S9-mediated mutagenicity was observed and when tested direct
247
mutagenicity was always reduced in the presence of $9. It is significant ( P 0.007), that the 41 dentures which produced mutagenic extracts were on the average worn for a longer period of time (mean 14.3 years) than the 28 mutagenically inactive dentures (mean 8.2 years). In this study, there was no statistical correlation between amount of tobacco usage and magnitude of mutagenic response, neither between mutagenicity and denture cleaning by soaking in cleansing solutions, denture repair i.e. relining and the consumption of alcoholic, coffee or tea beverages. Polymers such as PMMA-based macroreticular resins are well-known for their abilities to adsorb and reversibly bind small organic molecules in aqueous solutions (Dressier, 1979). This very property has been widely exploited in solid-phase extraction technology (Claxton et al., 1992; Schuetzle and Lewtas, 1986), including extraction and isolation of mutagenic substances from body fluids (Vennit, 1988). Denture base PMMA is not macroreticular in structure but it has a definite active surface and its noncrystalline structure may allow molecular diffusion into resin (Phillips, 1982). In addition, worn dentures are usually abraded and may be crazed and porosity is not an unusual structural defect, which may all lead to even more active surface area (Sweeney et al., 1955; Wolfaardt et al., 1986; Murray et al., 1986; Robinson et al., 1987; Arab et al., 1988). Thus, it is expected, albeit to a limited extent, that adsorption phenomena of this kind are bound to occur intra-orally. The fact that neither PMMA nor the methyl methacrylate monomer of acrylic dentures is mutagenic against TA98 or TA100 (Waegemaekers and Bensink, 1984; Thompson et al., 1991) indicates that the origin of denture extract mutagenicity is extraneous. It is, therefore, safe to infer that any of the dietary, tobacco and endogenous mutagenic substances were adsorbed onto acrylic dentures. Dietary mutagens include heterocyclic aromatic amines (Sugimura and Wakabayashi, 1990), polyaromatic hydrocarbons (Lijinsky, 1991), nitroso compounds (Tricker and Preussmann, 1991) and mutagenic flavonoids (MacGregor, 1986), furans and carbonyl com-
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pounds (Powrie et al., 1986). Tobacco mutagens include polyaromatic hydrocarbons, tobaccospecific and other nitrosamines and aromatic amines (DeMarini, 1983; Hecht and Hoffmann, 1988). Endogenous intra-oral mutagens may include in situ generated nitroso compounds (Hart and Walters, 1983) and perhaps microbially activated flavonoids (Parisis and Pritchard, 1983; Laires et al., 1989). Most of the above mutagens are active against both TA98 and TA100 (MacGregor, 1986; Sugimura and Wakabayashi, 1990; DeMarini, 1983; De Flora et al., 1984; Guttenplan, 1990). Direct mutagenicity is produced by some nitroso compounds (e.g. nitrosated phenols, indoles and amino-c~-carbolines) (Wakabayashi et al., 1989), flavonoids (MacGregor, 1986), carbony[ compounds (Powrie et al., 1986) and cigarettesmoke condensates (Curval et al., 1984). Mutagenicity of the denture extracts is likely to be due to a mixture of non-volatile mutagens (note evaporation methods) that possess both direct and indirect activity; however, it is impossible to assign this mutagenicity to any particular class of mutagens. The mutagenic difference against TA100 between smokers and non-smokers was probably due to a larger share of hydrophobic polyaromatic hydrocarbon-type of mutagens in smokers (De Flora et al., 1984; McCann et al., 1975) and the general predilection of activity against TA98 was to a large part probably due to mutagenically p o t e n t heterocyclic aromatic amines (Sugimura and Wakabayashi, 1990). Direct mutagenicity that was reduced by $9 is consistent with activity due to mutagenic carbonyl (Powrie et al., 1986) and direct-acting nitroso compounds (Wakabayashi et al., 1989). The denture extract mutagenic activity may only be an underestimate of the total denture mutagenicity as any existing volatile mutagens are not taken into account, the recovery of all non-volatile mutagens could not be absolutely quantitative and the possibility of antimutagenic interactions in the extract mixture (Dion and Bruce, 1983). Denture-adsorbed mutagens will have to desorb intra-orally in order to exert any effect. Frequent cycles of intra-orally high concentrations of ethanol in alcoholic beverages or mouthwashes may potentially mediate desorption, recirculation
and mucosal penetration of these mutagens (Squier et al., 1986; Smith et al., 1991; Horie et al., 1965; Winn et al., 1991). Such alcoholic desorptive regeneration could also counteract any beneficial in situ mutagen sequestering effect of dentures although saturation would have occurred at some point. Oral mucosal penetration by mutagens could be further enhanced by denture-induced atrophy, hypokeratinization, inflammation and ulceration (van Mens et al., 1975; Cook, 1991; Squier, 1991). Moreover, old, surface defective, ill-fitting dentures could increase levels of mutagenic adsorption and exposure with a possible superimposition of denture-induced traumatic effects and higher mitotic index of the basal and prickle cell layers (van Mens et al., 1975; Thumfart et al., 1978; Budtz-Jorgensen, 1981). This attains particular significance considering that there are over 20 million denture wearers in US and an estimated 30% of them feel they need refitting (Ad Hoc Committee, 1977); it has also been estimated that for the stabilization of ill-fitting dentures, over 7.5 million wearers use adhesive liners made of synthetic polymers including PMMA co-polymers (Shay, 1991; Jones ct al., 1991); other studies showed 50% of wearers suffer from chronic mucosal inflammation (Budtz-Jorgensen, 1981) and that most people over 65 are wearing dentures that are more than 10 years old (MacEntee, 1985). From a genotoxicological point of view, muta~ genicity of denture extracts comes at no surprise as the sensitive Ames test has already unveiled mutagenicity in human's most intimate environment (Vennit, 1988), including saliva (Stich et al., 1983; O'Connor et al., 1988) and oral mucosa scrapings (Parisis and Rao, 1986). The most important question then revolves around the biological significance of these oral mutagens especially in view of the multiple proto-oncogene and suppressor gene mutation basis of carcinogenesis (Barbacid, 1986; Boyd and Barrett, 1990) and the high predictivity of the Ames test (83% of detected mutagens are carcinogens) (Tennant eta[., 1987). Relevant are also the results of cytogenetic studies that showed quantitative variations in susceptibility to mutagen-induced genetic damage in oral cancer and cancer-free subjects (Schantz et
D.M. Parisis et al. /Mutation Research 321 (1994) 241-251
al., 1989) and the molecular biology studies which discovered point mutations of H-ras oncogene and inactivation of tumor suppressor genes as well as amplifications of H-, N- and K-ras, myc and erb-B genes in good proportions of oral cancer patients (Scully, 1992). While tobacco habits and alcohol overconsumption were established as the arch criminals consistently and independently influencing the causation of most oral cancers (Smith, 1989; Silverman Jr., 1990), many epidemiological studies also showed an association of denture wearing, poor-fitting dentures and non-specific denture irritation with oral cancer (Hoboek, 1949; Young et al., 1986; Winn, 1986). This association was generally discussed within the context of the relationship of persistent nonspecific oral trauma, poor oral hygiene and oral carcinogenesis (Thumfart et al., 1978; Smith, 1989). Nevertheless, one specifically designed epidemiological study using 400 oral cancer patients found no correlation between the wearing of dentures and any specific cancer site (Gorsky and Silverman Jr., 1984). It becomes evident that it is toxicologically important to systematically investigate oral mutagenic substances: their nature, origin, exposure levels, patterns of oral clearance and disposition, potential cancer initiating/promoting effects and the oral defense mechanisms against them (Perera, 1990). The presence of mutagens on dentures, for example, could provide a new "genotoxicological" hypothesis and a rationale to ascertain a measurable contributing role of denture wearing in a proportion of oro-pharyngeal cancers. The notion of toxic substances depositing on dentures, especially the older ones, may be used as an additional motive for the control of detrimental tobacco and alcohol habits, proper denture maintenance and replacement of old dysfunctional dentures and the overall advancement of the concept of good oral hygiene.
Acknowledgements We are indebted to Dr. Dan R. Leimann, of Hines V.A. Hospital Dental Clinics for his con-
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stant support and facilitation of the procurement of the denture specimens. The author (DMP) expresses his gratitude to Dr. Ioannis S. Scarpa, Dental Biochemistry Loyola University, for his encouragement and helpful discussions. We thank Drs. Dennis B. Solt and Christopher A. Squier for their critical reading of the manuscript.
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ison of liquid-liquid extraction and resin adsorption for concentrating mutagens in A m e s Salmonella/microsome assays on water, Bull. Environ. Contain. Toxicol., 27, 442449. Guttenplan, J.B. (1990) Mutagenesis by N-nitroso compounds: relationships to D N A adducts, D N A repair, and mutational efficiencies, Mutation Res., 233, 177-187. Hart, R.J., and C.L. Walters (1983) The formation of nitrite and N-nitroso compounds in salivas in vitro and in vivo, Fd. Chem. Toxicol., 21, 749 753. Hayatsu, H., K. Hamasaki, K. Togawa, S. Arimoto and T. Negishi (1983) Antimutagenic activity in extracts of h u m a n feces, in: H.F. Stich (Ed.), Carcinogens and Mutagens in the Environment, Vol. If, C R C Press, Boca Raton, Florida, pp. 91-99. Hecht, S.S., and D. Hoffmann (1988) Tobacco-specific nitrosamines, an important group of carcinogens in tobacco and tobacco smoke, Carcinogenesis, 9, 875-884. Hoboek, A. (1949) Dental prostheses and intraoral epidermoid carcinoma, Radiology, 44, 259-275. Horie, A., S. Kohchi and M. Kuratsune (1965) Carcinogenesis in the esophagus, II. Experimental production of esophageal cancer by administration of ethanolic solutions of carcinogens, Gann, 56, 429-441. Jones, D.W., G.C. Hall, E.J. Sutow, M.F. L a n g m a n and K.N. Robertson (1991) Chemical and Molecular weight analyses of prosthodontic soft polymers, J. Dent. Res., 70, 874-879. Khojasteh, A., and W.G. Kraybill (1988) Cancer of the esophagus: the environmental connection, Southern Med. J., 81, 878-882. Laires, A., P. Pacheco and J. Rueff (1989) Mutagenicity of rutin and the glycosidic activity of cultured cell-free microbial preparations of h u m a n faeces and saliva, Fd. Chem. Toxicol., 7, 437-443. Lijinsky, W. (1991) The formation and occurrence of polynuclear aromatic hydrocarbons associated with food, Mutation Res., 259, 251-261. MacEntee, M.I. (1985) The prevalence of edentulism and diseases related to dentures - - a literature review, J. Oral Rehab., 12, 195-207. MacGregor, J.T. (1986) Mutagenic and carcinogenic effects of flavonoids, in: V. Cody, E. Middleton Jr. and J.B. Harborne (Eds.), Plant Flavonoids in Biology and Medicine, Biochemical, Pharmacological and Structure-Activity Relationships, Liss, New York, pp. 411-424. Maron, D.M., and B.N. A m e s (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. McCann, J., E. Choi, E. Yamasaki and B.N. Ames (1975) D e t e c t i o n of c a r c i n o g e n s as m u t a g e n s in t h e Salmonella/microsome test: assay of 3(10 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72, 5135-5139. Murray, I.D., J.F. McCabe and R. Storer (1986) Abrasivity of denture cleaning pastes in vitro and in situ, Br. Dent. J., 161, 137-141. Nielsen, P.A. (1992) Mutagenicity studies on complex environmental mixtures: selection of solvent system for extraction, Mutation Res., 276, 117 123.
O'Connor, H.J., S.E. Riley, A.T.R. Axon, M.F. Dixon, A. Girling and R.C. Garner (1988) The mutagenic activity of gastric juice, Mutation Res., 206, 103-113. Parisis, D.M., and E.T. Pritchard (1983) Activation of rutin by h u m a n oral bacterial isolates to the carcinogen-mutagen quercetin, Arch. Oral Biol., 28, 583 590. Parisis, D.M., and G.S. Rao (1986) Mutagenicity in h u m a n oral mucosal scrapings, Fed. Proc. FASEB, 45, 441. Perera, F.P. (1990) Molecular epidemiology: a new tool in assessing risks of environmental carcinogens, CA-A Cancer J. Clin., 40, 277-288. Phillips, R.W. (1982) Science of Dental Materials, 8th edn., Saunders, Philadelphia. Powrie, W.D., C.H. Wu and V.P. Molund (1986) Browning reaction systems as sources of mutagens and antimutagens, Environ. Hlth. Perspect., 67, 47-54. Robinson, J.G., J.F. McCabe and R. Storer (1987) Denture bases: the effects of various treatments on clarity, strength and structure, J. Dent., 15, 159-165. Schantz, S.P., T.C. Hsu, N. Ainslie and R.P. Moser (1989) Young adults with head and neck cancer express increased susceptibility to mutagen-induced chromosome damage, J. Am. Med. Assoc., 262, 3313-3315. Schuetzle, D., and J. Lewtas (1986) Bioassay-directed chemical analysis in environmental research, Anal. Chem., 58, 1060 1075. Scully, C. (1992) Oncogenes, onco-suppressors, carcinogenesis and oral cancer, Br. Dent. J., 173, 53-59. Shay, K. (1991) Denture adhesives, J. Am. Dental Assoc., 122, 70-76. Silverman Jr., S. (1990) Oral Cancer, 3rd edn., American Cancer Society, Atlanta. Smith, C.J. (1989) Oral cancer and precancer: background, epidemiology and aetiology, Br. Dent. J., 167, 377-383. Smith, M.E., A.T. Cruchley and D.M. Williams (1991) The effect of ethanol on the penetration of benzo[a]pyrenc across h u m a n oral mucosa, J. Dent. Res., 7(/, 674. Squier, C.A. (1991) The permeability of oral mucosa, Crit. Rev. Oral Biol. Med., 2, 13-32. Squier, C.A., P. Cox and B.K. Hall (1986) Enhanced penetration of nitrosonornicotine across oral mucosa in the pres ence of ethanol, J. Oral Pathol., 15, 276-279. Stich, H.F., M.P. Rosin, C.H. Wu and W.D. Powrie (1982) The use of mutagenicity testing to evaluate food products, in: J.A. Heddle (Ed.), Mutagenicity in New Horizons in Genetic Toxicology, Academic Press, New York, pp. 117 142. Stich, H.F., B.A. Bohm, K. Chatterjee and J.L. Sailo (1983) The role of saliva-borne mutagens and carcinogens in the etiology of oral and esophageal carcinomas of betel nut and tobacco chewers, in: H.F. Stich (Ed.), Carcinogens and Mutagens in the Environment, Vol. 3, C R C Press, Boca Raton, FL, pp. 43-58. Sugimura, T., and K. Wakabayashi (1990) Mutagens and carcinogens in food, in: M.W. Pariza, J.S. Felton, H.-U. Aeschbacher and S. Sato (Eds.), Mutagens and Carcinogens in the Diet, Wiley-Liss, New York, pp. t-18.
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Sweeney, W.T., G.M. Brauer and I.C. Schoonover (1955) Crazing of acrylic resins, J. Dent. Res., 34, 306-312. Takeda, K., S. Ukawa and M. Mochizuki (1991) Inhibition by fatty acids of direct mutagenicity of N-nitroso compounds, in: I.K. O'Neill, J. Chen and H. Bartsch (Eds.), Relevance to Human Cancer of N-Nitroso Compounds, Tobacco and Mycotoxins, IARC Sci. Publ. No. 105, Lyon, France, pp. 558-563. Tennant, R.W., B.H. Margolin, M.D. Shelby, E. Zeiger, J.K. Haseman, J. Spalding, W. Caspary, M. Resnick, S. Stasiewicz, B. Anderson and R. Minor (1987) Prediction of chemical carcinogenicity in rodents from in vitro genetic toxicity assays, Science, 236, 933-941. Thompson, E.D., J.L. Seymour, M.J. Aardema, R.A. LeBoeuf, B.L.B. Evans and D.B. Cody (1991) Lack of genotoxicity of cross-linked acrylate polymers in 4 short-term genotoxicity assays, Environ. Mol. Mutagen., 18, 184-199. Thumfart, W., M. Weidenbecher, G. Waller and H.-J. Pesch (1978) Chronic mechanical trauma in the aetiology of oro-pharyngeal carcinoma, J. Max.-Fac. Surg., 6, 217-221. Tricker, A.R., and R. Preussmann (1991) Carcinogenic Nnitrosamines in the diet: occurrence, formation, mechanisms and carcinogenic potential, Mutation Res., 259, 277-289. van Mens, P.R., M.J. Pinkse-Veen and J. James (1975) Histological differences in the epithelium of denture-bearing and non-denture-bearing human palatal mucosa, Arch. Oral Biol., 20, 23-27.
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Vennit, S. (1988) The use of short-term tests for the detection of genotoxic activity in body fluids and excreta, Mutation Res., 205, 331-353. Waegemaekers, T.H.J.M., and M.P.M. Bensink (1984) Nonmutagenicity of 27 aliphatic acrylate esters in the Salmonella-microsome test, Mutation Res., 137, 95-102. Wakabayashi, K., M. Nagao and T. Sugimura (1989) Mutagens and carcinogens produced by the reaction of environmental aromatic compounds with nitrite, Cancer Surv., 8, 385-399. Winn, D.M. (1986) Smokeless tobacco and oral/pharynx cancer: the role of cofactors, in: D. Hoffmann and C.C. Harris (Eds.), Mechanisms in Tobacco Carcinogenesis, Banbury Report No. 23, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 361-375. Winn, D.M., W.J. Blot, J.K. McLaughlin, D.F. Austin, R.S. Greenberg, S. Preston-Martin, J.B. Schoenberg and J.F. Fraumeni Jr. (1991) Mouthwash use and oral conditions in the risk of oral and pharyngeal cancer, Cancer Res., 51, 3044-3047. Wolfaardt, J.E., P. Cleaton-Jones and P. Fatti (1986) The occurrence of porosity in a heat-cured poly(methyl methacrylate) denture base resin, J. Prosth. Dent., 55, 393-400. Young, T.B., C.N. Ford and J.H. Brandenburg (1986) An epidemiologic study of oral cancer in a statewide network, Am. J. Otolaryngol., 7, 200-208.
ELSEVIER
Mutation Research 321 (1994) 241-251
Mutagenicity of ethanolic extracts of used acrylic dentures D.M. Parisis a, N.L. Eskoz b, W.G. H e n d e r s o n c,. Department of Periodontics, Biochemistry and Microbiology, Loyola Unicersity, School of Dentistry, Maywood, IL, USA t~Dental Sercice, and c V.A. Cooperatil,e Studies Program, Hines V.A. Hospital, Hines, IL 60141-5151, USA (Received 14 April 1993; revision received 4 January 1994; accepted 6 January 1994)
Abstract The in vivo physicochemical sorption of mutagenic substances onto acrylic polymers was investigated in worn acrylic dentures. Thus, ethanolic extracts of acrylic dentures from 41 of a total of 69 human donors (60%), were found mutagenic in the standard plate incorporation Salmonella mutagenicity test against either TA98 or TA100 strains. Denture extracts from smokers produced mutagenicity more often than the ones from non-smokers (75% vs. 45%, P 0.01). Mutagenicity was preferentially directed against TA98 (TA98:TA100 = 2.9 : 1, P < 0.0005). Predilection for TA98 was more pronounced in denture extracts from non-smokers (4.7 : 1) than from smokers (2.0 : 1). When direct mutagenicity was observed, it was reduced by the rat-liver $9. Induced mutant yields were 6.1 + 3.9 and 7.0 _+ 8.9 times higher than the spontaneous for TA98 and TA100 respectively (smokers, 50-cm 2 denture surface area e q . / p l a t e + $9). Denture extracts from smokers induced higher levels of mutation than the ones from non-smokers (TA98 + $9, smoker:non-smoker = 2:1, P < 0.01). Mutagenicity was associated with longer periods of denture usage ( P 0.007). Thus, denture poly(methyl methacrylate) base material can adsorb mutagenic substances, possibly from diet and tobacco, which are extractable by ethanol. Theoretically, the in situ alcoholic desorption and recirculation of carcinogenic mutagens may have a contributory role in certain cases of intra-oral and upper alimentary tract carcinogenesis.
Key words: Acrylic dentures; Mutagen adsorption; Oro-pharyngeal carcinogenesis
1. Introduction
* Corresponding author: Dr. W.G. Henderson, c / o Dr. Dimitrios Parisis. This work was supported by the School of Dentistry, Loyola University of Chicago and Hines V.A. Hospital, Hines, IL, USA. AbbretJiations: HPLC, high-performance liquid chromatography; DMSO, (di)methyl sulfoxide; NPD, 4-nitro-l,2-phenylenediamine; B[o~]P, benzo[a]pyrene; PMMA, poly(methyl methacrylate). Elsevier Science B.V. SSDI 01 65-1 2 1 8 ( 9 4 ) 0 0 0 0 3 - L
A n i n t e g r a l p a r t o f the analysis of o r g a n i c m u t a g e n s in c o m p l e x m i x t u r e s is t h e i r e x t r a c t i o n a n d c o n c e n t r a t i o n (Claxton et al., 1992; S c h u e t z l e a n d Lewtas, 1986). T h e p r i n c i p l e o f s o r p t i o n o n t o p o r o u s o r g a n i c p o l y m e r s has b e e n successfully e x p l o i t e d ( D r e s s l e r , 1979) Thus, in c o m b i n a t i o n with m u t a g e n i c i t y assays, m u t a g e n analysis by s o r p t i o n o n t o A m b e r l i t e p o l y m e r i c a d s o r b e n t se-
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ries (e.g. X A D macroreticular acrylic resins) proved very fruitful in the detection and isolation of many organic mutagens in a variety of matrices (Grabow et al., 1981; Claxton et aI., 1992; Vennit, 1988). Acrylic denture base material is also a resin, a poly(methyl methacrylate). Denture resin has no macroreticular structure (Phillips, 1982), but otherwise it is chemically identical to XAD-8 Amberlite resin (Dressier, 1979) which is used for sorption-based analytical p r o c e d u r e s m e n t i o n e d above. It follows that orally transient mutagenic substances from tobacco smoke and diet for example, may become adsorbed onto the denture surface, resulting in retardation of their passage through the oral cavity. Their subsequent in situ desorption by alcohol may produce changed exposure patterns which might be a contributing factor in certain cases of oral and upper alimentary tract carcinogenesis (Stich et al., 1983; Khojasteh and Kraybill, 1988). The purpose of this study was to examine whether worn dentures from human subjects contain adsorbed mutagens that could be desorbed through the action of alcohol, by assessing the mutagenic activity of their ethanolic extracts in the S a l m o n e l l a / m i c r o s o m e mutagenicity test.
2. Materials and methods Chemicals
Ethyl alcohol-U.S.P. Dehydrated 200 Proof Punctilious was obtained from Q u a n t u m Chemical Corp., U.S.I. Division, Tuscola, IL. Methanol, m e t h y l e n e chloride and hexane, all high p u r i t y / H P L C grade, were from Burdick and Jackson Division of Baxter Healthcare Corp., Muskegon, MI. DMSO, H P L C grade and the diagnostic mutagens 4-nitro-l,2-phenylenediamine (NPD) and benzo[c~]pyrene (B[c~]P) were obtained from Aldrich Chemical Co., Milwaukee, WI. Agar was from Difco Laboratories, Detroit, M1 and Oxoid Nutrient Broth No. 2 from Oxoid LTD., Basingstoke, Hants., England. O t h e r chemicals and biochemicals were purchased from Sigma Chemical Co., St. Louis, MO, Mallinckrodt Inc., Paris, KY and J.T. Baker Chemical Co., Phillipsburg, NJ.
Table 1 Relevant data used from the history of denture donors Denture age (years worn) Denture-cleaning habits (cleansing solutions) Denture repair (relining) Tobacco-smokingstatus Alcohol consumption Coffee and/or tea consumption
Denture collection
Complete upper a n d / o r lower dentures were obtained from dental patients (donors) at Hines V.A. Hospital Dental Service Clinic. New dentures were made in exchange for the worn ones that were relinquished for use in this study. A brief patient history was taken with particular emphasis on items listed in Table 1. Collected dentures were wrapped in aluminum foil and stored at - 2 0 ° C until processed. Dentures from a total of 69 donors are included in this study: upper and lower dentures from 36 donors, upper denture from 31 donors and lower denture from 2 donors. Denture mutagen extraction
All procedures were performed in dim light to prevent breakdown of any photosensitive mutagens. Whenever donors contributed 2 denture pieces, both upper and lower dentures were pooled, processed and tested together. At no time were dentures from different donors ever mixed. First, the total surface area of each denture (both tissue fitting and non-fitting surfaces) was determined by covering with aluminum foil, tracing on standardized graph sheet (Keuffel and Esser Co., Morristown, N J) and weighing the cut-outs. Mean total surface area of pooled upper and lower dentures (n = 36), upper denture (n = 31) and lower denture (n = 2) was 174 _+ 24, 102 _+ 15 and 63 cm 2 respectively. Dentures were rinsed off with deionized water to free them from any gross debris. The denture(s) from each donor were then extracted with absolute ethanol, at 1 ml e t h a n o l / c m e denture surface area, in one-liter stoppered glass jars, by shaking in an incubator ( S / P Ultra-Tech Gold Series Shaking Water Bath BT-47, Baxter Healthcare Corp., McGaw Park, IL) at 37°C and 125 s t r o k e s / m i n for 1 h. This
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extraction was identically repeated 2 more times with fresh ethanol and the 3 successive extracts were pooled for each donor. The pooled ethanol extracts were passed through a 0.22 /~m MF-Millipore GS type filter (Millipore Sterifil Aseptic System, 47 mm) and then evaporated to dryness in a rotary evaporator (Yamato RE-51, Baxter) at 45°C. The residue was then redissolved in 1-1.5 ml of methanol which was then added to 30 ml of sodium phosphate buffer, 20 Mm, p H 7.0. This buffered mixture was then twice extracted with 2 volumes of hexane in a 250-mi separatory funnel using a Wrist Action Shaker, model 75 (Burrell Scientific Co., Pittsburgh, PA) at approximately 420 oscillations/min for 5 min. each cycle. These hexane extracts were discarded. The hexane preextraction step proved to limit the bacteriocidal effects of the extracts (hexane removes bacteriocidai and antimutagenic fatty acids with minimal extraction of pyrolysis mutagens) (Stich et al., 1982; Hayatsu et al., 1983; T a k e d a et al., 1991). The buffered mixture was subsequently extracted 3 times with 2 volumes of methylene chloride (Nielsen, 1992), under similar conditions. These extracts were pooled and saved. Then, the p H of the aqueous mixture was adjusted with N a O H to 10.0 and the methylene chloride extraction procedure was again applied (2 extractions). Finally, the p H of the mixture was readjusted with HC1 to 4.0 and the methylene chloride extraction procedure was repeated once again. Pooled methylene chloride extracts (for each donor) were combined and dried over N a z S O 4. The combined extracts were then rotary evaporated to dryness. The final residue was then redissolved in D M S O and the volume was adjusted to 1 /zl D M S O / c m 2 denture surface area. The residue was then assayed for mutagenicity in the A m e s test. The appearance and the net weight of the residues were routinely recorded.
Bacterial tester strains Salmonella typhimurium
strains TA98 and TA100 were kindly supplied by Dr. Bruce N. Ames of the University of California at Berkeley. These tester strains were grown, tested and maintained according to the revised methods of Maron and Ames (Maron and Ames, 1983).
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$9 fraction preparation Rat-liver $9 fraction was prepared based on the procedure of G a r n e r et al. (1972). Young male S p r a g u e - D a w l e y rats (Harlan S p r a g u e Dawley, Indianapolis, IN) weighing approximately 200 g each were used after induction with Aroclor 1254 (The Foxboro Co., Ultra Scientific, North Kingston, RI). The protein concentration of this $9 fraction was adjusted to 28 m g / m l and it was stored at - 8 0 ° C until used.
Mutagenicity assay The standard plate incorporation protocol of the Salmonella mutagenicity test was used without modification throughout this study (Maron and Ames, 1983). All procedures were also carried out in dim light. Briefly, denture extract residues corresponding to 25- and 50-cm 2 denture surface area in a total of 0.1 ml of D M S O were mixed with 0.1 ml of a standardized late exponential culture of tester strains grown the previous night (1-2 x 108 cells/plate) and 2 ml of top agar in the presence and in the absence of 0.5 ml of $9 mix (or sodium phosphate buffer, 0.1 M, pH 7.4) and poured onto minimal glucose plates. Due to the limited size of the individual dentures, the magnitude of mutagenic activity, testing against 2 strains sometimes in the absence of $9, the designed test concentrations of the extracts were not tested more than twice to produce S.D. The plates were incubated for 48 h in a 37°C dark incubator and then scored for revertant colonies. N P D ( 2 0 / z g / p l a t e ) and B[a]P (1 /xg/plate) were used in every assay (triplicates) as diagnostic mutagens. Extract concentrations with toxic (thinning o f / o r no background lawn) scores were re-. jected. No increase in the density of the bacterial lawn was ever observed to indicate interference by any histidine or its precursors in the final residue sample. A positive mutagenic response was considered when there was a doubling in the mutant yield (number of r e v e r t a n t s / p l a t e ) over the control without the extract residue.
Statistical analysis In comparing tester strains within the same individual dentures, M c N e m a r ' s chi-square test for correlated proportions was used. In making
D.M. Parisis et al. / Mutation Research 321 (1994) 241-251
244
comparisons of mutagenicity between smokers and non-smokers and other variables (denture cleaning habits, denture repair, alcohol, coffee and tea beverage consumption) the chi-square test for independent samples was used. In comparing mutant yield ratios between concentrations within each tester strain, the paired t-test was used. To compare mutant yield ratios between smokers and non-smokers and for the association of mutagenicity and degree of denture usage the t-test for independent samples was used. The relationship between pack-years of tobacco usage and mutant yield ratios was examined using Pearson's product-moment correlation coefficient. This relationship was also examined partialing out the effect of years of denture usage using the partial correlation coefficient.
3. Results
Table 2 Historic ~' spontaneous and induced mutant yields Tester
N umbe r of revertant c o l o n i e s / p l a t e
strain
-$9
+$9
TA100
146+23
142_+17
666+ 88
974+_22//
25+_ 6
39+_13
1680+162
254+_ 68
TA98
NPD h
Blot]P+ $9 c
" n = 12 assays. b 21) # g / p l a t e . " 1 ~zg/plate.
in the presence of $9 from 63 donors at extract concentrations corresponding to 50-cm 2 denture area e q . / p l a t e , except for the open symbols which correspond to 25-cm 2 eq./plate. 18 denture extracts within the non-smoker subgroup and 15 extracts within the smoker subgroup were tested against both strains (4 extracts from donors of questionable smoking status and 2 other extracts not tested with $9 are not shown). MYR of indi-
Results from dentures from a total of 69 donors are presented in this study. Mutagenicity is reported as the mutant yield ratio (MYR) of the experimental over the control, according to the formula:
28-27-/
1817-
D-( S9-sp ) MYR -
16-
sp
15I
where D is the mutant yield of the plates containing the denture extract residue, $9 the mutant yield of plates containing $9 mix plus D M S O and no extract and ap the spontaneous mutant yield of plates containing DMSO, buffer and neither $9 nor extract. MYR > 2 was considered a positive mutagenic response. Table 2 shows the historic spontaneous and NPD- and Bier]P-induced mutant yields of controis concurrently tested with the denture extracts. Because there was a significant association between mutagenicity and smoking status (denture extracts from 75% of smokers had MYR > 2 vs. 45% from non-smokers, P 0.01), results are shown separately for the smoker and non-smoker denture donor subgroups. Fig. 1 summarizes the results. It shows the mutant yield ratios produced by denture extracts
o 14--
rl- 1 3 - 12-11-10--
:E
9
--
8
--
7
--
6
--
5
--
4
!
•
I
I
I-
-i
3
I
--
2
TA100
TA98
Non-Smokers
TA100
TA98
Smokers
Fig. 1. Mutant yield ratios (extract:control) produced by denture extracts, in the presence of $9, from 63 donors. Extract concentrations correspond to 50-cm 2 denture area e q . / p l a t e except for the open symbols which correspond to 25-cm 2 e q . / p l a t e . For each strain only one concentration is shown.
D.M. Parisis et al. / M u t a t i o n Research 321 (1994) 241-251 Table 3 Percentage of donors with mutagenic denture extracts a Group (n = number of donors)
TA100
TA98
TA100 and/or TA98
23.1 (52)
76.5 (51)
59.4 (69)
Non-smoker
10.3 (29)
68.2 (22)
45.4 (33)
Smoker
42.1 (19)
78.6 (28)
75.0 (32)
29.4 (34)
85.3 (34)
-
Non-smoker
16.7 (18)
77.8 (18)
-
Smoker
46.7
93.3
-
(15)
(15)
GROUP I b All
G R O U P II c All
Tested with $9 (2 extracts tested against TA98 without $9) at concentrations corresponding to 25- a n d / o r 50-cm 2 denture surface area eq./plate. Mutagenic response considered positive when mutant yield ratio of experimental over control was greater than 2. h All the donors; individual extracts not necessarily tested against both strains; it also includes 4 donors of undetermined smoking status. TA100, non-smoker vs. smoker P 0.01; TA98, non-smoker vs. smoker P 0.4; TA100/TA98, non-smoker vs. smoker P 0.01. c Select donors from G R O U P I whose extracts were concurrently tested against both strains. All, TA100 vs. TA98 P < 0.0005; TA100, non-smoker vs. smoker P 0.05; TA98, nonsmoker vs. smoker P 0.18; non-smoker, TAI00 vs. TA98 P < 0.005; smoker, TA100 vs. TA98 P < 0.01.
vidual denture extracts ranged from 2.1 to 17.6 and 2.1 to 27.2 for TA98 and TA100 respectively. Overall, induced mutant yields reached a 6.1 _+ 3.9-fold and 7.0 + 8.9-fold increase over the spontaneous with TA98 and TA100 respectively (smokers, at extract concentration corresponding to 50-cm 2 denture surface area e q . / p l a t e ) . Table 3 shows the percentage of denture donors with mutagenic denture extracts against TA100 a n d / o r TA98, at 25- a n d / o r 50-cm 2 denture surface area e q . / p l a t e , in the presence of $9 (2 extracts were tested against TA98 in the absence of $9). G R O U P I represents all samples. Individual denture extracts were not necessarily
245
tested against both tester strains and it includes also 4 donors of undetermined smoking status. G R O U P II contains select donors from G R O U P I whose denture extracts were concurrently tested against both strains. Thus, denture extracts from 59.4% of the donors were found positive for mutagenicity. Results with 34 denture extracts that were concurrently tested against both strains ( G R O U P II) showed that extracts were more likely ( P < 0.0005) to be mutagenic against TA98 (85.3%) than against TA100 (29.4%) (TA98: TA100 ratio of 2.9:1). G R O U P I also showed a similar qualitative trend with 76.5% of them mutagenic against TA98 and 23.1% against TA100 (3.3 : 1). Within G R O U P I, denture extracts from smokers were more likely ( P 0.01) to be mutagenic on either strain (75.0%) than extracts from non-smokers (45.4%). This trend was particularly true ( P 0.01) with TA100 tester strain which showed a 4.1:1 smoker (42.1%) to non-smoker (10.3%) ratio of positive mutagenic response. The respective responses for smokers (78.6%) and non-smokers (68.2%) with TA98 showed no statistically significant difference between them ( P 0.4). Within each subgroup, results from denture extracts tested against both strains ( G R O U P II) showed a predilection for mutagenicity against TA98 a trend already observed with G R O U P I. Thus, extracts from 77.8% of non-smoker donors were mutagenic against TA98 as opposed to 16.7% of them that were mutagenic against TA100 ( P < 0.005). For the smoker subgroup the respective percentages were 93.3% for TA98 and 46.7% for TA100 ( P < 0.01). It is interesting to note that the intra-strain differences in the response between the 2 subgroups were greater with TA100 ( P 0.05 and a smoker to non-smoker ratio of 2.8: 1), than with TA98 ( P 0.18 and a respective ratio of 1.2: 1), reflecting a similar trend within G R O U P I. Table 4 shows the mutagenic potency of the denture extracts that were tested in the presence of $9 against TA100 a n d / o r TA98, at 2 5 - a n d / o r 50-cm 2 denture surface area e q . / p l a t e . G R O U P I represents all the mutagenic extracts from either non-smokers or smokers. Individual extracts were not necessarily tested against both strains nor at both concentrations. Thus, at the 50-cm 2
D.M. Par±sis et al. / M u t a t i o n Research 321 (1994) 241-251
246
Table 4 M u t a g e n i c i t y ~' o f d e n t u r e e x t r a c t s at 25- a n d 5 0 - c m 2 denture area eq./plate p l u s $9 Group (n = n u m b e r o f donors) GROUPI
TA100 25 c m 2
TA98 50 cm 2
25 c m 2
Non-smoker
4.9±4.4 (3)
2.2 (1)
3.0±1.3 (7)
3.1+0.6 (14)
Smoker
4.9±4.9 (5)
7.0±8.9 (7)
4.2±2.3 (14)
6.1+3.9 (17)
Smoker
Smoker
-
-
2.5 + 0 . 2
3.5±0.5
(6)
(6)
5.5+5.4
10.4+11.2
4.6± 2.4
7.1+4.3
(4)
(4)
(11)
(11)
5.5±5.4 (4)
7.4±9.7 (6)
5.9+3.6 (4)
7.4±5.2 (6)
III d
'~ Mutagenicity expressed as the mutant yield ratio of experimental ( d e n t u r e e x t r a c t ) o v e r c o n t r o l ( D M S O + $ 9 ) + SD. b All the mutagenic extracts (with $9, f r o m either non-smokers o r s m o k e r s ) ; individual dentures not necessarily tested against b o t h strains nor at b o t h c o n c e n t r a t i o n s . T A 9 8 at 25 c m 2, n o n - s m o k e r vs. s m o k e r P > 0.2; T A 9 8 at 50 c m 2, n o n - s m o k e r vs. s m o k e r P < 0.01. ~ M u t a g e n i c e x t r a c t s t e s t e d at b o t h concentrations against a particular strain. T A 9 8 n o n - s m o k e r o r s m o k e r , 25 c m 2 vs. 50 cm 2 P <0.01;TA100smoker, 2 5 c m 2 vs. 5 0 c m 2 P > 0 . 1 . d M u t a g e n i c e x t r a c t s c o n c u r r e n t l y tested against both strains at the same c o n c e n t r a t i o n . A t 25 c m 2 o r 50 c m z, T A 1 0 0 vs. T A 9 8 P > 0.8.
eq. concentration and with TA98, potency was higher (P < 0.01) in the smoker subgroup (1.96 : 1 smoker to non-smoker ratio). There are no adequate data with the non-smoker subgroup tested against TA100 at this concentration for a similar comparison. G R O U P II represents the mutagenic extracts that were tested at both concentrations against a particular strain. There was a dose-response relationship of potency in each subgroup with TA98. Thus, the 50-cm 2 eq.:25-cm 2 eq. dose ratio of MYR was 1.4:1 for the nonsmoker subgroup ( P < 0.01) and 1.5:1 for the smoker subgroup (P < 0.01). G R O U P III represents the mutagenic extracts (from smokers) that were concurrently tested against both strains at the same concentration. Thus, when MYR for the
E x t r a c t No.
- $9
+ $9
TAI00
l
3.3
2.2
TA98
2 3 4 5 6 7 8 9 10
2.3 2.5 3.0 < 2.0 < 2.0 < 2.0 2.7 9.2 15.8
2.1 2.3 7.0 2.5 7.0 11).5
c
Non-smoker
GROUP
Mutagenicity ~' o f select b d e n t u r e e x t r a c t s t h a t were tested in the presence a n d / o r in the absence of the $9 fraction Tester strain
50 c m 2
b
GROUPII
Table 5
" Mutagenicity as in T a b l e 4 ± $9. i, E x t r a c t s t h a t when tested with a n d / o r w i t h o u t $9 w e r e either directly a n d / o r indirectly m u t a g e n i c ; all f r o m smokers (except No. 8); all t e s t e d at 2 5 - c m 2 denture surface area eq./plate ( e x c e p t No. 7 a n d No. 8, t e s t e d at the 5 0 - c m 2 eq. concentration).
same concentration were compared no differences were found between the 2 tester strains (P > 0.8 for each concentration). In Table 5 shows that certain extracts when appropriately tested exhibited direct mutagenic activity or S9-mediated mutagenicity. All 4 extracts that were both directly and indirectly mutagenic yielded decreased mutagenic activity in the presence of the $9 metabolic system. Table 6 shows that positive mutagenic response was statistically correlated with the degree of denture usage (P 0.007). There was no significant relationship between pack-years and magnitude of mutagenic response even after partialing out the effect of denture usage. For TA98, the correlation coefficient of pack-years and mutagenic response at 25 cm: was r - 0 . 1 7 (n = 16, P 0.58) and at 50 cm 2 was
Table 6 R e l a t i o n o f mutagenic activity a of denture e x t r a c t s to the n u m b e r o f y e a r s o f denture usage
Mutagenic activity
Years + S D
(n = n u m b e r o f donors)
Negative (28) Positive (41) " Positive w h e n MYR > 2. Negative vs. positive P 0.007.
8 . 2 + 6.2 14.3 ± 11.5
D.M. Parisis et a l . / Mutation Research 321 (1994) 241-251
r - 0 . 0 0 3 (n = 18, P 0.99). After partialing out the effect of years worn, the partial correlation coefficient was r 0.09 ( P 0.81) and r 0.22 ( P 0.57) for 25 and 50 cm 2 respectively. For TA100 the corresponding coefficients were r - 0 . 1 3 (n = 7 , P 0.78) at 25 cm 2 and r - 0 . 0 6 ( n = 7 , P 0.91) at 50 cm 2. After partialing out, the partial coefficients were r 0.41 ( P 0.5) and r 0.43 ( P 0.47) respectively. No statistically significant correlation was found between mutagenicity and denture cleaning habits ( P 0.474), denture repair ( P 0.135), alcoholic beverage consumption ( P 0.845) and coffee or tea drinking ( P 0.85).
4. Discussion
This study reveals that a significant percentage of our denture donors (60%) wore dentures whose methylene chloride-fractionated ethanolic extracts contained substances mutagenic towards the Salmonella tester strains TA98 and TA100. Denture extracts from smokers were more likely to be found mutagenic, against either strain, than the ones from non-smokers (75% vs. 45%, P 0.01); however, when frequency of occurrence was measured separately for each strain, this relationship attained statistical significance with TA100 strain only (smoker 42% vs. non-smoker 10%, P 0.01). Mutagenicity was preferentially manifested with the frame-shift mutation detector strain TA98 than with the base-pair mutation detector strain TA100 (TA98:TA100 = 2.9 : 1, P < 0.0005). This predilection for TA98 was more pronounced in the non-smoker subgroup (4.7:1, P < 0.005) than in the smoker subgroup (2.0 : 1, P < 0.01). Mutagenic potency was 2 times higher in smokers than in non-smokers (TA98, at 50-cm 2 denture surface area equivalent per plate and in the presence of metabolic activation, P < 0.01). Overall, induced mutant yields were 6.1 _+ 3.9 and 7.0_+ 8.9 times over the spontaneous for TA98 and TA100 respectively (smoker denture donors, at 50-cm 2 denture surface area e q . / p l a t e + $9) Both direct and rat-liver S9-mediated mutagenicity was observed and when tested direct
247
mutagenicity was always reduced in the presence of $9. It is significant ( P 0.007), that the 41 dentures which produced mutagenic extracts were on the average worn for a longer period of time (mean 14.3 years) than the 28 mutagenically inactive dentures (mean 8.2 years). In this study, there was no statistical correlation between amount of tobacco usage and magnitude of mutagenic response, neither between mutagenicity and denture cleaning by soaking in cleansing solutions, denture repair i.e. relining and the consumption of alcoholic, coffee or tea beverages. Polymers such as PMMA-based macroreticular resins are well-known for their abilities to adsorb and reversibly bind small organic molecules in aqueous solutions (Dressier, 1979). This very property has been widely exploited in solid-phase extraction technology (Claxton et al., 1992; Schuetzle and Lewtas, 1986), including extraction and isolation of mutagenic substances from body fluids (Vennit, 1988). Denture base PMMA is not macroreticular in structure but it has a definite active surface and its noncrystalline structure may allow molecular diffusion into resin (Phillips, 1982). In addition, worn dentures are usually abraded and may be crazed and porosity is not an unusual structural defect, which may all lead to even more active surface area (Sweeney et al., 1955; Wolfaardt et al., 1986; Murray et al., 1986; Robinson et al., 1987; Arab et al., 1988). Thus, it is expected, albeit to a limited extent, that adsorption phenomena of this kind are bound to occur intra-orally. The fact that neither PMMA nor the methyl methacrylate monomer of acrylic dentures is mutagenic against TA98 or TA100 (Waegemaekers and Bensink, 1984; Thompson et al., 1991) indicates that the origin of denture extract mutagenicity is extraneous. It is, therefore, safe to infer that any of the dietary, tobacco and endogenous mutagenic substances were adsorbed onto acrylic dentures. Dietary mutagens include heterocyclic aromatic amines (Sugimura and Wakabayashi, 1990), polyaromatic hydrocarbons (Lijinsky, 1991), nitroso compounds (Tricker and Preussmann, 1991) and mutagenic flavonoids (MacGregor, 1986), furans and carbonyl com-
248
D.M. Parisis et al. / Mutation Research 321 (1994) 241-251
pounds (Powrie et al., 1986). Tobacco mutagens include polyaromatic hydrocarbons, tobaccospecific and other nitrosamines and aromatic amines (DeMarini, 1983; Hecht and Hoffmann, 1988). Endogenous intra-oral mutagens may include in situ generated nitroso compounds (Hart and Walters, 1983) and perhaps microbially activated flavonoids (Parisis and Pritchard, 1983; Laires et al., 1989). Most of the above mutagens are active against both TA98 and TA100 (MacGregor, 1986; Sugimura and Wakabayashi, 1990; DeMarini, 1983; De Flora et al., 1984; Guttenplan, 1990). Direct mutagenicity is produced by some nitroso compounds (e.g. nitrosated phenols, indoles and amino-c~-carbolines) (Wakabayashi et al., 1989), flavonoids (MacGregor, 1986), carbony[ compounds (Powrie et al., 1986) and cigarettesmoke condensates (Curval et al., 1984). Mutagenicity of the denture extracts is likely to be due to a mixture of non-volatile mutagens (note evaporation methods) that possess both direct and indirect activity; however, it is impossible to assign this mutagenicity to any particular class of mutagens. The mutagenic difference against TA100 between smokers and non-smokers was probably due to a larger share of hydrophobic polyaromatic hydrocarbon-type of mutagens in smokers (De Flora et al., 1984; McCann et al., 1975) and the general predilection of activity against TA98 was to a large part probably due to mutagenically p o t e n t heterocyclic aromatic amines (Sugimura and Wakabayashi, 1990). Direct mutagenicity that was reduced by $9 is consistent with activity due to mutagenic carbonyl (Powrie et al., 1986) and direct-acting nitroso compounds (Wakabayashi et al., 1989). The denture extract mutagenic activity may only be an underestimate of the total denture mutagenicity as any existing volatile mutagens are not taken into account, the recovery of all non-volatile mutagens could not be absolutely quantitative and the possibility of antimutagenic interactions in the extract mixture (Dion and Bruce, 1983). Denture-adsorbed mutagens will have to desorb intra-orally in order to exert any effect. Frequent cycles of intra-orally high concentrations of ethanol in alcoholic beverages or mouthwashes may potentially mediate desorption, recirculation
and mucosal penetration of these mutagens (Squier et al., 1986; Smith et al., 1991; Horie et al., 1965; Winn et al., 1991). Such alcoholic desorptive regeneration could also counteract any beneficial in situ mutagen sequestering effect of dentures although saturation would have occurred at some point. Oral mucosal penetration by mutagens could be further enhanced by denture-induced atrophy, hypokeratinization, inflammation and ulceration (van Mens et al., 1975; Cook, 1991; Squier, 1991). Moreover, old, surface defective, ill-fitting dentures could increase levels of mutagenic adsorption and exposure with a possible superimposition of denture-induced traumatic effects and higher mitotic index of the basal and prickle cell layers (van Mens et al., 1975; Thumfart et al., 1978; Budtz-Jorgensen, 1981). This attains particular significance considering that there are over 20 million denture wearers in US and an estimated 30% of them feel they need refitting (Ad Hoc Committee, 1977); it has also been estimated that for the stabilization of ill-fitting dentures, over 7.5 million wearers use adhesive liners made of synthetic polymers including PMMA co-polymers (Shay, 1991; Jones ct al., 1991); other studies showed 50% of wearers suffer from chronic mucosal inflammation (Budtz-Jorgensen, 1981) and that most people over 65 are wearing dentures that are more than 10 years old (MacEntee, 1985). From a genotoxicological point of view, muta~ genicity of denture extracts comes at no surprise as the sensitive Ames test has already unveiled mutagenicity in human's most intimate environment (Vennit, 1988), including saliva (Stich et al., 1983; O'Connor et al., 1988) and oral mucosa scrapings (Parisis and Rao, 1986). The most important question then revolves around the biological significance of these oral mutagens especially in view of the multiple proto-oncogene and suppressor gene mutation basis of carcinogenesis (Barbacid, 1986; Boyd and Barrett, 1990) and the high predictivity of the Ames test (83% of detected mutagens are carcinogens) (Tennant eta[., 1987). Relevant are also the results of cytogenetic studies that showed quantitative variations in susceptibility to mutagen-induced genetic damage in oral cancer and cancer-free subjects (Schantz et
D.M. Parisis et al. /Mutation Research 321 (1994) 241-251
al., 1989) and the molecular biology studies which discovered point mutations of H-ras oncogene and inactivation of tumor suppressor genes as well as amplifications of H-, N- and K-ras, myc and erb-B genes in good proportions of oral cancer patients (Scully, 1992). While tobacco habits and alcohol overconsumption were established as the arch criminals consistently and independently influencing the causation of most oral cancers (Smith, 1989; Silverman Jr., 1990), many epidemiological studies also showed an association of denture wearing, poor-fitting dentures and non-specific denture irritation with oral cancer (Hoboek, 1949; Young et al., 1986; Winn, 1986). This association was generally discussed within the context of the relationship of persistent nonspecific oral trauma, poor oral hygiene and oral carcinogenesis (Thumfart et al., 1978; Smith, 1989). Nevertheless, one specifically designed epidemiological study using 400 oral cancer patients found no correlation between the wearing of dentures and any specific cancer site (Gorsky and Silverman Jr., 1984). It becomes evident that it is toxicologically important to systematically investigate oral mutagenic substances: their nature, origin, exposure levels, patterns of oral clearance and disposition, potential cancer initiating/promoting effects and the oral defense mechanisms against them (Perera, 1990). The presence of mutagens on dentures, for example, could provide a new "genotoxicological" hypothesis and a rationale to ascertain a measurable contributing role of denture wearing in a proportion of oro-pharyngeal cancers. The notion of toxic substances depositing on dentures, especially the older ones, may be used as an additional motive for the control of detrimental tobacco and alcohol habits, proper denture maintenance and replacement of old dysfunctional dentures and the overall advancement of the concept of good oral hygiene.
Acknowledgements We are indebted to Dr. Dan R. Leimann, of Hines V.A. Hospital Dental Clinics for his con-
249
stant support and facilitation of the procurement of the denture specimens. The author (DMP) expresses his gratitude to Dr. Ioannis S. Scarpa, Dental Biochemistry Loyola University, for his encouragement and helpful discussions. We thank Drs. Dennis B. Solt and Christopher A. Squier for their critical reading of the manuscript.
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