0099-2399/92/1811-0527/$03,00/0 JOURNAL OF ENDODONTICS Copyright © 1992 by The American Association of Endodontists
Printed in U.S.A.
VOL. 18, NO. 11, NOVEMBER1992
SCIENTIFIC ARTICLES Catalase/Peroxidase Activity in Dental Pulp William H. Bowles, PhD, DDS, and Henry Burns, Jr.
Extrinsic stains on vital teeth are bleached with 30% hydrogen peroxide (H=O2) or carbamide peroxide, H20= greatly inhibits the activity of several enzymes. Free H=O2 and carbamide peroxide readily enter the pulp through the coronal wall of the tooth. Nevertheless, adverse effects have been remarkably rare. This study was undertaken to determine whether dental pulp exhibits any catalase or peroxidase activity that might protect it from damage during vital bleaching procedures. PuIpal tissue from healthy human teeth was assayed for catalase and glutathione peroxidase activity. A phosphate buffer extract of the tissue served as the source of the enzymes. The rate of breakdown of H202 by the tissue extract was measured and the rate constant for catalase was determined. The catalase activity, defined as ,M H202 broken down/rain/rag wet tissue, was determined and found to be only 2 x 10 -=, which is very low. The fibrous pulpai tissue was found to exhibit virtually no glutathione peroxidase activity.
enzymes studied were inhibited to some degree by hydrogen peroxide, some were completely inactivated. When the enzyme preparations were treated with various concentrations of hydrogen peroxide at 50"C, only two enzymes retained as much as 50% of their original activity; two other enzymes retained only 20 to 25% activity and three others were reduced to 0 to 8% of original activity. Hoffmann and Meneghini (7) demonstrated that hydrogen peroxide is toxic to cultured human fibroblasts in concentrations as low as 10 ~zM (0.34 mg/liter), as judged by loss of colony-forming ability, which they showed to be due to single strand breaks in DNA. Bowles and Ugwuneri (8) showed that hydrogen peroxide readily penetrates the coronal walls of freshly extracted human teeth to enter the pulp chamber and that the rate of diffusion appeared to double for each 10" rise in temperature, and speculated regarding the "Qlo rule" for chemical processes. The recent increase in the use of carbamide peroxide bleaching agents prompted the investigation of the ability of these agents to penetrate the dental hard tissue to reach the pulp chamber (9). It was shown that exposure to 10% carbamide peroxide caused significant quantities of hydrogen peroxide to diffuse into the pulp chambers of freshly extracted teeth, and pulpal peroxide increased with increased carbamide peroxide concentration. Given the sensitivity of pulpal enzymes and nuclear DNA to hydrogen peroxide, it is surprising that there have been so few reported instances of pulpitis and pulpal necrosis. Robertson and Melfi (2), Seale et al. (4), and Zach and Cohen (5) all suggest that pulp tissue possesses marvelous recovery ability. One possible mechanism by which the pulp may protect itself from damage by H202 is by enzymatic breakdown of the molecule. Hydrogen peroxide may he degraded by two classes of enzymes: peroxidase and catalase. Peroxidase uses hydrogen peroxide to oxidize some other substrate, while catalase breaks down hydrogen peroxide to water and oxygen. The purpose of this study was to determine whether pulpal tissue exhibits any peroxide-degrading enzyme activity.
Concentrated hydrogen peroxide has been used for many years to safely and effectively bleach extrinsic dental stains, with only rare instances of transient inflammation or sensitivity (1-4). Often the process was augmented by application of a heat source. Externally applied heat is difficult to control and the temperature to which the pulp is subjected cannot be directly determined. Zach and Cohen (5) placed tiny thermister probes in teeth of rhesus monkeys, applied a heat source to the labial surface, and measured the time required to raise the pulpal temperature by 4°F, 10*F, 20*F, or 30*F. They then applied the heat source to the contralateral teeth for the same time intervals, removed the teeth, and examined the pulps histologically. They found that all teeth in the groups in which the temperature was increased by 4 and 10*F eventually recovered. Thirteen of 20 teeth elevated by 20*F recovered, whereas none of the teeth in the 30*F category recovered. Bowles and Thompson (6) examined the effects of hydrogen peroxide alone and, with applied heat, on the activity of a number of pulpal enzymes. They found that most of the
MATERIALS AND METHODS Commercial 30% hydrogen peroxide (Superoxol; Merck, Rahway, N J) was standardized against K_IV[nO4,which had in turn been standardized against dried sodium oxalate (10). A
527
528
B o w l e s and Burns
standard curve was made for H202 based on the absorbance of H202 at 240 nm, with concentrations ranging from 0.015 mM to 0.15 raM. Caries-free human third molars were obtained at time of extraction and packed in ice. The teeth were fractured and the pulps were extirpated, combined, and weighed. The pulpal tissues were placed in cold phosphate buffer (50 mM, pH 7), containing 10% by volume of a 0.25% sodium cholate solution and homogenized in a glass homogenizer. The homogenate was centrifuged at 5"C and the supernatant decanted. Cellular debris was resuspended and washed in 2 ml of cold phosphate buffer, recentrifuged, and the wash combined with the original supernatant, and made to a volume of 25 ml. The pulpal extract was assayed for catalase activity according to the method described by Aebi (11). H202 absorbs UV light at 240 nm in direct proportion to concentration. Therefore, the rate of decrease in absorbance can be used as a measure of decrease in substrate concentration, to assay a tissue extract for catalase activity. At relatively low concentrations of H202, the enzymatic decomposition follows a firstorder reaction rate and the rate constant is equal to the enzyme concentration. A blank was prepared containing 2.0 ml of tissue homogenate (enzyme) and 1 ml of distilled water. Samples were prepared by pipetting 2.0 ml of enzyme and 2.0 ml of working peroxide solution, (30 mM) into a test tube which was capped with Parafilm and the contents quickly mixed by inversion. The mixture was transferred to a cuvette and placed in the spectrophotometer at a wave length setting of 240 nm. Changes in absorbance were read at 15-s intervals for a period of 3 rain. Absorbance readings were proportional to the quantity of hydrogen peroxide broken down and were compared with the standard curve for conversion to tzmol H202. Concentration of hydrogen peroxide was plotted versus time in minutes. Glutathione (GSH) peroxidase is another enzyme that plays a major role in the detoxification of hydrogen peroxide. The activity of this enzyme was measured to determine whether it plays a role in the breakdown of hydrogen peroxide in dental pulp. The assay was performed according to the method described by Floh6 and Giinzler (12). These authors discussed the complexities of the kinetics of GSH peroxidase. In this procedure GSH peroxidase is measured indirectly by coupling two reactions together, as follows:
Journal of Endodontics
of standardized 30% H202. The pulpal extract served as the source of the enzyme. The following solutions were combined in a semi-micro cuvette (1 ml): 500 ~1 of phosphate buffer, 100 tzl of enzyme (tissue homogenate extract), 100 #1 of GSH reductase (0.24 units), and 100 tzl of 10.0 mM GSH. The resulting solution was mixed thoroughly and equilibrated at 37"C for 10 min. Then 100 #1 of NADPH solution were added and the change in absorbance at 340 nm was recorded for 3 min against a blank with no NADPH. This measured the nonhydrogen peroxide-dependent consumption of NADPH. Then 100 ul ofH202, prewarmed to 37"C, were added and quickly mixed. The decrease in absorbance was monitored for 5 min. RESULTS In a first-order enzymatically catalyzed reaction the rate constant, k, is a measure of the enzyme activity, according to the following equation (11): k = (2.3/At) (log
where At = t2 - t, = the measured time interval and S, and $2 = H202 concentrations at times t~ and t2. The breakdown of hydrogen peroxide by catalase is shown in Fig. 1. The rate constant was calculated from data presented in Fig. 1, according to the above equation: k = (2.3/3) (log 65.4/48.2) = 0.102 umol/min. When the weight of the original tissue and the dilution factor of the solution were accounted for, pulpal catalase activity was found to be only 2 x 10-2 umol/min/mg of fresh tissue. This is very low, based on information derived from Aebi (11), which indicates that the k for catalase is on the order of 105 umol/min. Glutathione peroxidase activity was found to be negligible (Fig. 2). DISCUSSION Peroxidase activity has been examined in neutrophils and eosinophils (13) and in serum (14). Serum activity was con-
)eroxide concentration (micromols)
1. GSH + H202 --~ GSSG + 2 H20
7O
2. GSSG + NADPH --~ 2 GSH + NADP+
so
Reaction i is catalyzed by GSH peroxidase, while reaction 2 is catalyzed by GSH reductase. In this coupled reaction, GSH is maintained at a constant level while hydrogen peroxide is broken down. The conversion of NADPH to NADP ÷ is readily followed by spectrophotometrically monitoring the change in absorbance at 340 nm as NADPH is oxidized, providing an indirect measure of GSH peroxidase activity. GSH reductase (yeast) was obtained from Sigma Chemical Co. (St. Louis, MO) and diluted with phosphate buffer (pH 7.0) according to manufacturer's directions, to a concentration of 2.4 units/ml. An aqueous solution of GSH (10 mM) was prepared. The concentration of this solution is critical. A 1.5 mM solution of NADPH was prepared in 0.1% sodium bicarbonate. A H202 solution (1.5 mM) was made by dilution
S~/$2)
40
SO 20
10 0
I
I
I
~
I
I
0.5
1
1.5
2
2.5
3
3.5
time in minutes m
Seriee 1
FIG 1. Rate of decomposition of hydrogen peroxide by pulpal catalase
activity showing the change in peroxide concentration versus time in minutes.
Pulpal Peroxidase Activity
VoI. 18, NO. 11, November 1992 0.07 0.06
amm|mawmm|m|mm|m~mmm|
| ~ m a |
0.05 I 0.04 0.03 0.02
!
0"01 I 0
I
I
I
I
I
0.2
0.4
0.6
0.8
1
1.2
Time in minutes ="-
Series 2
FiG 2. GSH peroxidase activity is shown to be absent in dental pulpal extract.
sidered to be related to neutrophil turnover. Dental pulpal detoxification of hydrogen peroxide has apparently not been previously investigated. Pulp tissue from extracted teeth exhibits very little entrapment of blood, so that pulpal enzyme activity was not thought to be significantly influenced by neutrophil or eosinophil peroxidase activity. However, during inflammation, the larger volumes of blood flowing through the pulp could contribute a greater amount of peroxidase activity. Dental pulp has a rather sparse cell population composed primarily of fibroblasts, with a large volume of extracellular connective tissue, which undoubtedly contributes to the low enzyme activity. There is a conspicuous lack of relief mechanisms in dental pulp. Encasement in hard tissue precludes swelling in the presence of inflammation and hyperemia, with a consequent increase in hydrostatic pressure and odontalgia. In a case report, Glickman, et al. (15) treated a patient who presented with an abscessed maxillary central incisor. The tooth was nonvital and had been asymptomatic until an attempt was made to bleach it, following which the abscess resulted. This case tends to confirm reports of laboratory studies demonstrating the penetration of peroxides into the pulp chamber (6, 8). Quantities of peroxides reported to penetrate the pulp chamber of extracted teeth are equal to or greater than the quantities reported to produce toxic effects in cultured fibro-
529
blasts (7). These studies, and studies reporting the inhibition of pulpal enzymes by hydrogen peroxide (5), along with the present study demonstrating the lack of peroxidase activity in dental pulp all suggest the need for caution in the use of peroxide bleaching agents, especially by individuals not under the direct supervision of a dentist. In spite of these reports there are amazingly few reports of major untoward responses to the use of dental bleaching agents. Careful follow-up studies regarding the incidence and severity of hypersensity in postbleaching cases might prove enlightening. It is noteworthy that the U.S. Food and Drug Administration has recently ruled that tooth-whitening agents are considered as drugs and now require a New Drug Application before they can be marketed as tooth-whitening agents. Dr. Bowles and Mr. Bums are members of the Department of Biochemistry, Baylor College of Dentistry, Dallas, TX. Address requests for reprints to Dr. William Bowles, Department of Biochemistry, Baylor College of Dentistry, 3302 Gaston Avenue, Dallas, TX 75246.
References 1. Murrin JR, Barkemeier WW. Chemical treatment of endemic dental fluorosis. Quint tnt 1982;13:363-9. 2. Robertson WD, Melfi RC. Pulpal responses to vital bleaching procedures. J Endodon 1980;6:645-9. 3. Cohen SC. Human pulpal response to bleaching procedures on vital teeth. J Endodon 1979;5:134-8. 4. Seale NS, Mclntosh JE, Taylor AN. Pulpal reaction to bleaching of teeth in dogs. J Dent Res 1981;60:948-53. 5. Zach L, Cohen G. Pulp response to externally applied heat. Oral Surg 1965;19:515-30. 6. Bowles WH, Thompson LR. Vital bleaching: the effect of heat and hydrogen peroxide on pulpal enzymes. J Endodon 1986;12:108-12. 7. Hoffmann ME, Meneghini R. Action of hydrogen peroxide on human fibroblasts Jn culture. Photochem Photobiol 1979;30:151-5. 8. Bowles WH, Ugwuneri Z. Pulp chamber penetration by hydrogen peroxide following vital bleaching procedures. J Endodon 1987;13:375-7. 9. Cooper JS, Bokmeyer T J, Bowles WH. Penetration of the pulp chamber by carbamide peroxide bleaching agents. J Endodon (in press). 10. United States Pharmacopeia XVII. Easton, PA: Mack Publishing Co., 1965;991, 1086. 11. Aebi H. Catalase in vitro. In: Packer L, ed. Methods in Enzymology. 1984;105:121-126. 12. Floh6 L, G0nzler WA. Assays of glutathione peroxidase, in: Packer L, ed. Methods in enzymology. Vo1105, 1984:114-21. 13. Kitahara M, Simonian Y, Eyre HJ. Neutrophil myeloperoxidase: a simple, reproducible technique to determine activity. J Lab ClJn Med 1979;93:232-7. 14. Venge P, Foucard T, Henriksen J, Hakansson L, Kreuger A. Serum levels of lactoferrin, lysozyme and myeloperoxidase in normal, infection-prone and leukemic children. Clin Chim Acta 1984;136:121-30. 15. Glickman GN, Frysh H, Baker FL Adverse periadicular response to vital bleaching. J Endodon (in press).
Printed in U.S.A.
VOL. 18, NO. 11, NOVEMBER1992
SCIENTIFIC ARTICLES Catalase/Peroxidase Activity in Dental Pulp William H. Bowles, PhD, DDS, and Henry Burns, Jr.
Extrinsic stains on vital teeth are bleached with 30% hydrogen peroxide (H=O2) or carbamide peroxide, H20= greatly inhibits the activity of several enzymes. Free H=O2 and carbamide peroxide readily enter the pulp through the coronal wall of the tooth. Nevertheless, adverse effects have been remarkably rare. This study was undertaken to determine whether dental pulp exhibits any catalase or peroxidase activity that might protect it from damage during vital bleaching procedures. PuIpal tissue from healthy human teeth was assayed for catalase and glutathione peroxidase activity. A phosphate buffer extract of the tissue served as the source of the enzymes. The rate of breakdown of H202 by the tissue extract was measured and the rate constant for catalase was determined. The catalase activity, defined as ,M H202 broken down/rain/rag wet tissue, was determined and found to be only 2 x 10 -=, which is very low. The fibrous pulpai tissue was found to exhibit virtually no glutathione peroxidase activity.
enzymes studied were inhibited to some degree by hydrogen peroxide, some were completely inactivated. When the enzyme preparations were treated with various concentrations of hydrogen peroxide at 50"C, only two enzymes retained as much as 50% of their original activity; two other enzymes retained only 20 to 25% activity and three others were reduced to 0 to 8% of original activity. Hoffmann and Meneghini (7) demonstrated that hydrogen peroxide is toxic to cultured human fibroblasts in concentrations as low as 10 ~zM (0.34 mg/liter), as judged by loss of colony-forming ability, which they showed to be due to single strand breaks in DNA. Bowles and Ugwuneri (8) showed that hydrogen peroxide readily penetrates the coronal walls of freshly extracted human teeth to enter the pulp chamber and that the rate of diffusion appeared to double for each 10" rise in temperature, and speculated regarding the "Qlo rule" for chemical processes. The recent increase in the use of carbamide peroxide bleaching agents prompted the investigation of the ability of these agents to penetrate the dental hard tissue to reach the pulp chamber (9). It was shown that exposure to 10% carbamide peroxide caused significant quantities of hydrogen peroxide to diffuse into the pulp chambers of freshly extracted teeth, and pulpal peroxide increased with increased carbamide peroxide concentration. Given the sensitivity of pulpal enzymes and nuclear DNA to hydrogen peroxide, it is surprising that there have been so few reported instances of pulpitis and pulpal necrosis. Robertson and Melfi (2), Seale et al. (4), and Zach and Cohen (5) all suggest that pulp tissue possesses marvelous recovery ability. One possible mechanism by which the pulp may protect itself from damage by H202 is by enzymatic breakdown of the molecule. Hydrogen peroxide may he degraded by two classes of enzymes: peroxidase and catalase. Peroxidase uses hydrogen peroxide to oxidize some other substrate, while catalase breaks down hydrogen peroxide to water and oxygen. The purpose of this study was to determine whether pulpal tissue exhibits any peroxide-degrading enzyme activity.
Concentrated hydrogen peroxide has been used for many years to safely and effectively bleach extrinsic dental stains, with only rare instances of transient inflammation or sensitivity (1-4). Often the process was augmented by application of a heat source. Externally applied heat is difficult to control and the temperature to which the pulp is subjected cannot be directly determined. Zach and Cohen (5) placed tiny thermister probes in teeth of rhesus monkeys, applied a heat source to the labial surface, and measured the time required to raise the pulpal temperature by 4°F, 10*F, 20*F, or 30*F. They then applied the heat source to the contralateral teeth for the same time intervals, removed the teeth, and examined the pulps histologically. They found that all teeth in the groups in which the temperature was increased by 4 and 10*F eventually recovered. Thirteen of 20 teeth elevated by 20*F recovered, whereas none of the teeth in the 30*F category recovered. Bowles and Thompson (6) examined the effects of hydrogen peroxide alone and, with applied heat, on the activity of a number of pulpal enzymes. They found that most of the
MATERIALS AND METHODS Commercial 30% hydrogen peroxide (Superoxol; Merck, Rahway, N J) was standardized against K_IV[nO4,which had in turn been standardized against dried sodium oxalate (10). A
527
528
B o w l e s and Burns
standard curve was made for H202 based on the absorbance of H202 at 240 nm, with concentrations ranging from 0.015 mM to 0.15 raM. Caries-free human third molars were obtained at time of extraction and packed in ice. The teeth were fractured and the pulps were extirpated, combined, and weighed. The pulpal tissues were placed in cold phosphate buffer (50 mM, pH 7), containing 10% by volume of a 0.25% sodium cholate solution and homogenized in a glass homogenizer. The homogenate was centrifuged at 5"C and the supernatant decanted. Cellular debris was resuspended and washed in 2 ml of cold phosphate buffer, recentrifuged, and the wash combined with the original supernatant, and made to a volume of 25 ml. The pulpal extract was assayed for catalase activity according to the method described by Aebi (11). H202 absorbs UV light at 240 nm in direct proportion to concentration. Therefore, the rate of decrease in absorbance can be used as a measure of decrease in substrate concentration, to assay a tissue extract for catalase activity. At relatively low concentrations of H202, the enzymatic decomposition follows a firstorder reaction rate and the rate constant is equal to the enzyme concentration. A blank was prepared containing 2.0 ml of tissue homogenate (enzyme) and 1 ml of distilled water. Samples were prepared by pipetting 2.0 ml of enzyme and 2.0 ml of working peroxide solution, (30 mM) into a test tube which was capped with Parafilm and the contents quickly mixed by inversion. The mixture was transferred to a cuvette and placed in the spectrophotometer at a wave length setting of 240 nm. Changes in absorbance were read at 15-s intervals for a period of 3 rain. Absorbance readings were proportional to the quantity of hydrogen peroxide broken down and were compared with the standard curve for conversion to tzmol H202. Concentration of hydrogen peroxide was plotted versus time in minutes. Glutathione (GSH) peroxidase is another enzyme that plays a major role in the detoxification of hydrogen peroxide. The activity of this enzyme was measured to determine whether it plays a role in the breakdown of hydrogen peroxide in dental pulp. The assay was performed according to the method described by Floh6 and Giinzler (12). These authors discussed the complexities of the kinetics of GSH peroxidase. In this procedure GSH peroxidase is measured indirectly by coupling two reactions together, as follows:
Journal of Endodontics
of standardized 30% H202. The pulpal extract served as the source of the enzyme. The following solutions were combined in a semi-micro cuvette (1 ml): 500 ~1 of phosphate buffer, 100 tzl of enzyme (tissue homogenate extract), 100 #1 of GSH reductase (0.24 units), and 100 tzl of 10.0 mM GSH. The resulting solution was mixed thoroughly and equilibrated at 37"C for 10 min. Then 100 #1 of NADPH solution were added and the change in absorbance at 340 nm was recorded for 3 min against a blank with no NADPH. This measured the nonhydrogen peroxide-dependent consumption of NADPH. Then 100 ul ofH202, prewarmed to 37"C, were added and quickly mixed. The decrease in absorbance was monitored for 5 min. RESULTS In a first-order enzymatically catalyzed reaction the rate constant, k, is a measure of the enzyme activity, according to the following equation (11): k = (2.3/At) (log
where At = t2 - t, = the measured time interval and S, and $2 = H202 concentrations at times t~ and t2. The breakdown of hydrogen peroxide by catalase is shown in Fig. 1. The rate constant was calculated from data presented in Fig. 1, according to the above equation: k = (2.3/3) (log 65.4/48.2) = 0.102 umol/min. When the weight of the original tissue and the dilution factor of the solution were accounted for, pulpal catalase activity was found to be only 2 x 10-2 umol/min/mg of fresh tissue. This is very low, based on information derived from Aebi (11), which indicates that the k for catalase is on the order of 105 umol/min. Glutathione peroxidase activity was found to be negligible (Fig. 2). DISCUSSION Peroxidase activity has been examined in neutrophils and eosinophils (13) and in serum (14). Serum activity was con-
)eroxide concentration (micromols)
1. GSH + H202 --~ GSSG + 2 H20
7O
2. GSSG + NADPH --~ 2 GSH + NADP+
so
Reaction i is catalyzed by GSH peroxidase, while reaction 2 is catalyzed by GSH reductase. In this coupled reaction, GSH is maintained at a constant level while hydrogen peroxide is broken down. The conversion of NADPH to NADP ÷ is readily followed by spectrophotometrically monitoring the change in absorbance at 340 nm as NADPH is oxidized, providing an indirect measure of GSH peroxidase activity. GSH reductase (yeast) was obtained from Sigma Chemical Co. (St. Louis, MO) and diluted with phosphate buffer (pH 7.0) according to manufacturer's directions, to a concentration of 2.4 units/ml. An aqueous solution of GSH (10 mM) was prepared. The concentration of this solution is critical. A 1.5 mM solution of NADPH was prepared in 0.1% sodium bicarbonate. A H202 solution (1.5 mM) was made by dilution
S~/$2)
40
SO 20
10 0
I
I
I
~
I
I
0.5
1
1.5
2
2.5
3
3.5
time in minutes m
Seriee 1
FIG 1. Rate of decomposition of hydrogen peroxide by pulpal catalase
activity showing the change in peroxide concentration versus time in minutes.
Pulpal Peroxidase Activity
VoI. 18, NO. 11, November 1992 0.07 0.06
amm|mawmm|m|mm|m~mmm|
| ~ m a |
0.05 I 0.04 0.03 0.02
!
0"01 I 0
I
I
I
I
I
0.2
0.4
0.6
0.8
1
1.2
Time in minutes ="-
Series 2
FiG 2. GSH peroxidase activity is shown to be absent in dental pulpal extract.
sidered to be related to neutrophil turnover. Dental pulpal detoxification of hydrogen peroxide has apparently not been previously investigated. Pulp tissue from extracted teeth exhibits very little entrapment of blood, so that pulpal enzyme activity was not thought to be significantly influenced by neutrophil or eosinophil peroxidase activity. However, during inflammation, the larger volumes of blood flowing through the pulp could contribute a greater amount of peroxidase activity. Dental pulp has a rather sparse cell population composed primarily of fibroblasts, with a large volume of extracellular connective tissue, which undoubtedly contributes to the low enzyme activity. There is a conspicuous lack of relief mechanisms in dental pulp. Encasement in hard tissue precludes swelling in the presence of inflammation and hyperemia, with a consequent increase in hydrostatic pressure and odontalgia. In a case report, Glickman, et al. (15) treated a patient who presented with an abscessed maxillary central incisor. The tooth was nonvital and had been asymptomatic until an attempt was made to bleach it, following which the abscess resulted. This case tends to confirm reports of laboratory studies demonstrating the penetration of peroxides into the pulp chamber (6, 8). Quantities of peroxides reported to penetrate the pulp chamber of extracted teeth are equal to or greater than the quantities reported to produce toxic effects in cultured fibro-
529
blasts (7). These studies, and studies reporting the inhibition of pulpal enzymes by hydrogen peroxide (5), along with the present study demonstrating the lack of peroxidase activity in dental pulp all suggest the need for caution in the use of peroxide bleaching agents, especially by individuals not under the direct supervision of a dentist. In spite of these reports there are amazingly few reports of major untoward responses to the use of dental bleaching agents. Careful follow-up studies regarding the incidence and severity of hypersensity in postbleaching cases might prove enlightening. It is noteworthy that the U.S. Food and Drug Administration has recently ruled that tooth-whitening agents are considered as drugs and now require a New Drug Application before they can be marketed as tooth-whitening agents. Dr. Bowles and Mr. Bums are members of the Department of Biochemistry, Baylor College of Dentistry, Dallas, TX. Address requests for reprints to Dr. William Bowles, Department of Biochemistry, Baylor College of Dentistry, 3302 Gaston Avenue, Dallas, TX 75246.
References 1. Murrin JR, Barkemeier WW. Chemical treatment of endemic dental fluorosis. Quint tnt 1982;13:363-9. 2. Robertson WD, Melfi RC. Pulpal responses to vital bleaching procedures. J Endodon 1980;6:645-9. 3. Cohen SC. Human pulpal response to bleaching procedures on vital teeth. J Endodon 1979;5:134-8. 4. Seale NS, Mclntosh JE, Taylor AN. Pulpal reaction to bleaching of teeth in dogs. J Dent Res 1981;60:948-53. 5. Zach L, Cohen G. Pulp response to externally applied heat. Oral Surg 1965;19:515-30. 6. Bowles WH, Thompson LR. Vital bleaching: the effect of heat and hydrogen peroxide on pulpal enzymes. J Endodon 1986;12:108-12. 7. Hoffmann ME, Meneghini R. Action of hydrogen peroxide on human fibroblasts Jn culture. Photochem Photobiol 1979;30:151-5. 8. Bowles WH, Ugwuneri Z. Pulp chamber penetration by hydrogen peroxide following vital bleaching procedures. J Endodon 1987;13:375-7. 9. Cooper JS, Bokmeyer T J, Bowles WH. Penetration of the pulp chamber by carbamide peroxide bleaching agents. J Endodon (in press). 10. United States Pharmacopeia XVII. Easton, PA: Mack Publishing Co., 1965;991, 1086. 11. Aebi H. Catalase in vitro. In: Packer L, ed. Methods in Enzymology. 1984;105:121-126. 12. Floh6 L, G0nzler WA. Assays of glutathione peroxidase, in: Packer L, ed. Methods in enzymology. Vo1105, 1984:114-21. 13. Kitahara M, Simonian Y, Eyre HJ. Neutrophil myeloperoxidase: a simple, reproducible technique to determine activity. J Lab ClJn Med 1979;93:232-7. 14. Venge P, Foucard T, Henriksen J, Hakansson L, Kreuger A. Serum levels of lactoferrin, lysozyme and myeloperoxidase in normal, infection-prone and leukemic children. Clin Chim Acta 1984;136:121-30. 15. Glickman GN, Frysh H, Baker FL Adverse periadicular response to vital bleaching. J Endodon (in press).
