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Effects of lipopolysaccharides on human dental pulp cells
0099-2399/95/2103-0128/$03.00/0 JOURNAL OF ENDOOONTICS Copyright © 1995 by The American Association of Endodontists
Printed in U.S.A. VOL. 21, No. 3, MARCH1995
Effects of Lipopolysaccharides on Human Dental Pulp Cells Akinobu Nakane, DDS, PhD, Tsutomu Yoshida, DDS, Kazuhiko Nakata, DDS, PhD, Naoki Horiba, DDS, PhD, and Hiroshi Nakamura, DDS, PhD
Human dental pulp cells were treated with 1, 10, and 100 ~g/ml of lipopolysaccharide (LPS). The effects of treatment were examined by measurement of the DNA content, protein content, and alkaline phosphatase activity of the cells. LPS samples were purified from Porphyromonas gingivalis, Porphyromonas endodontalis, and Fusobacterium nucleatum isolated from root canals, and Escherichia coil 0111:B4 LPS was used as a positive control. At a concentration of 1 ~g/ml, none of the LPSs caused any change in the production of DNA or protein, whereas the amount of DNA was increased at 10 ~g/ml and inhibited at 100 ~g/ml. Protein synthesis was decreased by LPSs at both 10 and 100 ~g/ml. Alkaline phosphatase activity was not changed at any concentration of LPS tested.
gg 4~
a
3 2 r~ 1 0 /zg
o j
It is well known that bacteria are one of the most important causative agents ofpulpitis. Many bacteria have been isolated from infected root canals. Hashioka et al. (1) reported that there is a relationship between clinical symptoms and anaerobic bacteria from the infected root canal. Many anaerobes (such as Peptococcus, Peptostreptococcus,Eubacterium, Pro-
/zg
"/
iO
i4 days
9
lb
h days
4"
C
-3"
pionibacteriurn, Lactobacillus, Veillonella, Fusobacterium, Porphyromonas, Bacteroides, and others) have been isolated
z].
from infected canals. The Gram-negative ones (Veillonella,
0
Fusobacterium, Porphyromonas, and Bacteroides) contain a
Pg
lipopolysaccharide (LPS) complex. Pinero et al. (2) reported that a high level of LPS was obviously toxic to connective tissue containing fibroblasts and the extracellular matrix, and would result in tissue necrosis. There are a few reports that have evaluated the correlation between LPS and periapical lesions. Horiba et al. (3) indicated a positive correlation between LPS and clinical symptoms or a radiolucent area. However, no investigations have evaluated the effect of LPS on human dental pulp cells. The purpose of the present study was to evaluate the effect of LPS on human dental pulp ceils by measurement of the DNA content, protein content, and alkaline phosphatase activity of the cells.
3
7
10
4,
14 days
d
3
0
i
i
1'0
1'4 days
FIG 1. Effect of E. coli (a), P. gingivalis (b), ,0. endodontalis (c), and F. nucleatum (d) LPS on DNA production by human pulp cells, e, control; O, 1 #g/ml of LPS; A, 10 #g/ml of LPS; I-I, 100 #g/ml of LPS. Values are means + SD. * p < 0.05; ** p < 0.01.
128
~lfel. 21, No. 3, March 1995 •
X
LPS on Dental Pulp Cells
102pg
129
mU/pg DNA
I0
× 1023
7
10
14 days
0
i
mU/B g DNA
i
I'0
1'4 days
")
1"0
1'4 days
b
t
2
< 5]
X ]02p g
")
1'0
F
8 6
1'4 days C
.,
0
20! 25"
.~
15"
4
¢~ lOI
2
< 5"
0
× 102p3~;
7
10
14 days
•
mU/pIDNA
0
C
J
3 mU//.tg DNA 25.~2oI
7
10
14 days
d
I0' <
0
.~
7
1'0
1'4 days
FIG 2. Effect of E. coli (a),P. gingiva/is (b), P. endodonta/is (c),and F. nucleatum (d) LPS on protein production by human pulp cells,e, control; O, 1 #g/ml of LPS; 4, 10 #g/ml of LPS; I-I, 100/~g/ml of LPS. Values are means _+ SD. * p < 0.05; ** p < 0.01.
5'
.~
"I
l'O
1'4 days
FIG 3. Effect of E. coli (a), P. gingivalis (b), P. endodontalis (c), and F. nucleatum (d) LPS on alkaline phosphatase (ALPase) activity of human pulp cells, e, control; O, 1 #g/ml of LPS; A, 10/~g/ml of LPS; 1"7,100 #g/ml of LPS. Values are means _+ SD.
MATERIALS AND METHODS A freshly extracted human tooth was washed with sterile saline solution containing antibiotics (I000 units/ml of penicillin and 30 ~g/ml of fungizone). The tooth was cut along its longitudinal axis to obtain the pulp. Once the pulpal tissue had been washed with Eagle's minimal essential medium (MEM) containing antibiotics (100 units/ml of penicillin, 100 ug/ml of streptomycin, and 3 ~g/ml of fungizone), a small amount (1.5 x 1.5 mm) was placed in a culture dish (35 mm diameter) with 10% fetal bovine serum (FBS)-Eagle's MEM and incubated at 37"C in an atmosphere of 5% CO2/95% air until a confluent cell monolayer had formed. The human dental pulp cells were used after four to eight subcultures. LPS samples were prepared from Porphyromonas gingivalis AEI2, Porphyromonas endodontalis AE51, and Fusobacteriurn nucleatum AE91 isolated from infected root canals of patients seen at the Department of Endodontics, School of Dentistry, Aichi Gakuin University. Escherichia coli 0111 :B4
LPS was used as a positive control. The LPS from each type of bacterium was extracted and purified by the hot phenolwater procedure of Westphal et al. (4). Human dental pulp cells (2 ml; 2.5 x 104 cells/ml) were seeded in culture dishes (35 mm diameter) in MEM supplemented with 10% FBS. After incubation for 24 h at 37"C in 5% CO2/95% air atmosphere, the cell monolayer was washed two times with calcium- and magnesium-free, phosphatebuffered saline solution. Thereafter, 2 ml of 10% FBS-MEM containing 1, 10, or 100 #g/ml of LPS was added to each of five dishes. Further incubation was done for 3 to 14 days; the DNA content, protein content, and alkaline phosphatase activity in cells were measured. The DNA content was measured by a modification of the method of Hinegardner (5); protein content, with a Bio-Rad Protein Assay kit (6); and alkaline phosphatase activity, by the procedure of Bessey et al. (7). All statistical evaluations were made with Student's t test.
130
Journal of Endodontics
Nakane et al.
RESULTS Production of DNA by pulpal cells was increased significantly by E. coil LPS at 10 ug/ml at 10 and 14 days (p < 0.01), and inhibited at 100 ~g/ml at all experimental periods (p < 0.01); increased by P. gingivalis LPS at 10 #g/ml at 3, 7, and 10 days (p < 0.05, p < 0.01) and inhibited by it at 100 ug/ml at 3 and 7 days (p < 0.01); decreased by P. endodontalis LPS at 100 #g/ml at 3 and 7 days (p < 0.01); and increased by F. nucleatum LPS at 10 jzg/ml at 3 and 7 days (p < 0.05) and decreased by it at 100 ug/ml at 3, 7, and 10 days (p < 0.01 ) (Fig. 1). E. coli LPS at 10 #g/ml inhibited pulp cell protein production significantly at 3 and 7 days (p < 0.01) and at 100 ug/ ml, at all experimental periods (p < 0.01); P. gingivalis LPS at 100 t~g/ml blocked it at 3, 7, and 10 days (p < 0.01); P. endodontalis LPS at 100 #g/ml, at all experimental periods (p < 0.01); and F. nucleatum LPS at 100 #g/ml, at 3, 7, and 10 days (p < 0.01) (Fig. 2). Alkaline phosphatase activity was unaffected by any LPS at any concentration throughout the experimental period (Fig. 3).
when the pulp is inflamed. However, there was no effect of any LPS on the differentiation state of human dental pulp cells. Schein and Schilder (11), Dahlen and Bergenholtz (12), and Horiba et al. (13) reported that the average value of the LPS content detected in the infected root canal ranged from 1 to 100 ~g/ml. Therefore the concentration of 1 to 100 ug/ ml of LPS was used in this study. It is known that the LPS has a cytotoxic effect on various cells, such as lymphocytes, human periodontal ligament-derived fibroblasts, and others, and stimulates them to produce cytokines like interleukin-l, interleukin-6, tumor necrosis factors, and so forth (14-16). These cytokines are mediators of inflammation. It would be very worthwhile to investigate the relationship between LPS and the release of cytokines from human dental pulp cells. Drs. Nakane, Yoshida, Nakata, Horiba, and Nakamura are members of the Department of Endodontics, School of Dentistry, Aichi Gakuin University, Nagoya, Japan. Address requests for reprints to Dr. Akinobu Nakane, Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya 464, Japan.
References
DISCUSSION Several reports have investigated a correlation between periapical disease and LPS content. Yamasaki et al. (8) reported a positive correlation between the development of periapical lesions and an increase in LPS content. However, few investigations have shown a correlation between the breakdown of pulp matrix and LPS content. Pinero et al. (2) reported that, when the uptake of [3H]thymidine by bovine pulp fibroblast cultures was measured in the cell fraction at 6, 24, and 48 h, there was an increase over the control when the cells were treated with 5 ug/ml of LPS purified from E. coil, no change with 125 ~g/ml, and an inhibitory effect with 625 ~g/ml. Isomura (9) reported that the LPS from F. nucleaturn showed cytotoxic effects on HeLa cells with an LDso concentration of 489 #g/ml. Also, Sugizaki (10) calculated the concentration of LPS for 50% inhibition of the control to be 280 t~g/ml. Our results indicate that production of DNA in human dental pulp cells was inhibited significantly at 100 ~g/ml LPS purified from E. coli at all experimental periods, thus differing from the findings of Pinero et al. (2). With respect to the sensitivity of cells to LPS, differences in the origin of the cells as well as differences in culture conditions may be important factors. Also, the discrepancy could be caused by the difference in LPS preparation. Production of protein in human dental pulp cells was inhibited at 10 ttg/ml of LPS from E. coil and was inhibited slightly at I0 #g/ml of LPS from P. gingivalis, P. endodontalis, and F. nucleatum. Our results showed that 10 ~g/ml of E. coli LPS provoked an increase in the production of DNA and a decrease in that of protein. The reason for this finding may be caused by a difference between the molecular structure of E. coli LPS and that of others. The alkaline phosphatase activity was measured to evaluate the effect of LPS on the differentiation ability of the cells. Large amounts of alkaline phosphatase are found in pulpal odontoblasts when the cells are active in calcification and
1. Hashioka K, Yamasaki M, Nakane A, Hodbe N, Nakamura H. The relationship between clinical symptoms and anaerobic bacteria from infected root canals. J Endodon 1992;18:558-61. 2. Pinero G, Kiatpongsah S, Hutchins MO, Hoover J. The effect of endotoxin on the synthesis of connective tissue matrix components by pulp fibroblasts in vitro. J Endodon 1983;9:2-7. 3. Hodba N, Maekawa Y, Abe Y, Ito M, Matsumoto T, Nakamura H. Correlations between endotoxin and clinical symptoms or radiolucent areas in infected root canals. Oral Surg 1991 ;71:492-5. 4. Westphal O, Jann K. Extraction with phenol-water and further applications of the procedure. Methods Carbohydrate Chem 1965;5:83-91. 5. Hinegardner RT. An improved fluorometric assay for DNA. Anal Biochem 1971 ;39:197-201. 6. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54. 7. Bessey OA, Lowry OH, Brock MJ. A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J Biol Chem 1946; 164:321-9. 8. Yamasaki M, Nakane A, Kumazawa M, Hashioka K, Hodbe N, Nakamura H. Endotoxin and gram-negative bacteria in the rat pedapical lesions. J Endodon 1992;18:501-4. 9. Isomura K. Studies on bacterial endotoxin from Fusobacterium nucleaturn--especially the role of endotoxin in periodontal diseases. J Jpn So(: Perodont 1985;27:328-51 (in Japanese). 10. Sugizaki K. Metabolic alteration of Gin-1 flbroblast by lipopolysaccharide from pedodontopathic bacteda. J Japan Soc Periodont 1986;28:468-83 (in Japanese). 11. Schein B, Schilder H. Endotoxin content in endodontically involved teeth. J Endodon 1975;1:19-21. 12. Dahlen G, Bergenholtz G. Eodotoxic activity in teeth with necrotic pulps. J Dent Res 1980;59:1033-40. 13. Horiba N, Maekawa Y, Matsumoto T, Nakamura H. A study of the distdbution of endotoxin in the dentinal wall of infected root canals. J Endodon 1990; 16:331-4. 14. Hameda S, Takada H, Ogawa T, Fujiwara T, Mihara J. Upopolysaccharides of oral anaerobes associated with chronic inflammation: chemical and immunomodulating properties. Intern Rev Immuno11990;6:247-61. 15. Takada T, Mihara J, Modsaki I, Hamada S. Induction of intedeukin-1 and -6 in human gingival fibroblast cultures stimulated with Bacteroides [ipopolysaccharides. Infect Immun 1991 ;59:295-301. 16. Yamazaki K, Ikarashi F, Aoyagi T, Takahashi K, Nakajima T, Hara K, Seymour GJ. Direct and indirect effects of Porphyromonas gingivalis lipopotysaccharide on intedeukin-6 production by human gingival fibroblasts. Oral Microbio11992;7:218-24.
Printed in U.S.A. VOL. 21, No. 3, MARCH1995
Effects of Lipopolysaccharides on Human Dental Pulp Cells Akinobu Nakane, DDS, PhD, Tsutomu Yoshida, DDS, Kazuhiko Nakata, DDS, PhD, Naoki Horiba, DDS, PhD, and Hiroshi Nakamura, DDS, PhD
Human dental pulp cells were treated with 1, 10, and 100 ~g/ml of lipopolysaccharide (LPS). The effects of treatment were examined by measurement of the DNA content, protein content, and alkaline phosphatase activity of the cells. LPS samples were purified from Porphyromonas gingivalis, Porphyromonas endodontalis, and Fusobacterium nucleatum isolated from root canals, and Escherichia coil 0111:B4 LPS was used as a positive control. At a concentration of 1 ~g/ml, none of the LPSs caused any change in the production of DNA or protein, whereas the amount of DNA was increased at 10 ~g/ml and inhibited at 100 ~g/ml. Protein synthesis was decreased by LPSs at both 10 and 100 ~g/ml. Alkaline phosphatase activity was not changed at any concentration of LPS tested.
gg 4~
a
3 2 r~ 1 0 /zg
o j
It is well known that bacteria are one of the most important causative agents ofpulpitis. Many bacteria have been isolated from infected root canals. Hashioka et al. (1) reported that there is a relationship between clinical symptoms and anaerobic bacteria from the infected root canal. Many anaerobes (such as Peptococcus, Peptostreptococcus,Eubacterium, Pro-
/zg
"/
iO
i4 days
9
lb
h days
4"
C
-3"
pionibacteriurn, Lactobacillus, Veillonella, Fusobacterium, Porphyromonas, Bacteroides, and others) have been isolated
z].
from infected canals. The Gram-negative ones (Veillonella,
0
Fusobacterium, Porphyromonas, and Bacteroides) contain a
Pg
lipopolysaccharide (LPS) complex. Pinero et al. (2) reported that a high level of LPS was obviously toxic to connective tissue containing fibroblasts and the extracellular matrix, and would result in tissue necrosis. There are a few reports that have evaluated the correlation between LPS and periapical lesions. Horiba et al. (3) indicated a positive correlation between LPS and clinical symptoms or a radiolucent area. However, no investigations have evaluated the effect of LPS on human dental pulp cells. The purpose of the present study was to evaluate the effect of LPS on human dental pulp ceils by measurement of the DNA content, protein content, and alkaline phosphatase activity of the cells.
3
7
10
4,
14 days
d
3
0
i
i
1'0
1'4 days
FIG 1. Effect of E. coli (a), P. gingivalis (b), ,0. endodontalis (c), and F. nucleatum (d) LPS on DNA production by human pulp cells, e, control; O, 1 #g/ml of LPS; A, 10 #g/ml of LPS; I-I, 100 #g/ml of LPS. Values are means + SD. * p < 0.05; ** p < 0.01.
128
~lfel. 21, No. 3, March 1995 •
X
LPS on Dental Pulp Cells
102pg
129
mU/pg DNA
I0
× 1023
7
10
14 days
0
i
mU/B g DNA
i
I'0
1'4 days
")
1"0
1'4 days
b
t
2
< 5]
X ]02p g
")
1'0
F
8 6
1'4 days C
.,
0
20! 25"
.~
15"
4
¢~ lOI
2
< 5"
0
× 102p3~;
7
10
14 days
•
mU/pIDNA
0
C
J
3 mU//.tg DNA 25.~2oI
7
10
14 days
d
I0' <
0
.~
7
1'0
1'4 days
FIG 2. Effect of E. coli (a),P. gingiva/is (b), P. endodonta/is (c),and F. nucleatum (d) LPS on protein production by human pulp cells,e, control; O, 1 #g/ml of LPS; 4, 10 #g/ml of LPS; I-I, 100/~g/ml of LPS. Values are means _+ SD. * p < 0.05; ** p < 0.01.
5'
.~
"I
l'O
1'4 days
FIG 3. Effect of E. coli (a), P. gingivalis (b), P. endodontalis (c), and F. nucleatum (d) LPS on alkaline phosphatase (ALPase) activity of human pulp cells, e, control; O, 1 #g/ml of LPS; A, 10/~g/ml of LPS; 1"7,100 #g/ml of LPS. Values are means _+ SD.
MATERIALS AND METHODS A freshly extracted human tooth was washed with sterile saline solution containing antibiotics (I000 units/ml of penicillin and 30 ~g/ml of fungizone). The tooth was cut along its longitudinal axis to obtain the pulp. Once the pulpal tissue had been washed with Eagle's minimal essential medium (MEM) containing antibiotics (100 units/ml of penicillin, 100 ug/ml of streptomycin, and 3 ~g/ml of fungizone), a small amount (1.5 x 1.5 mm) was placed in a culture dish (35 mm diameter) with 10% fetal bovine serum (FBS)-Eagle's MEM and incubated at 37"C in an atmosphere of 5% CO2/95% air until a confluent cell monolayer had formed. The human dental pulp cells were used after four to eight subcultures. LPS samples were prepared from Porphyromonas gingivalis AEI2, Porphyromonas endodontalis AE51, and Fusobacteriurn nucleatum AE91 isolated from infected root canals of patients seen at the Department of Endodontics, School of Dentistry, Aichi Gakuin University. Escherichia coli 0111 :B4
LPS was used as a positive control. The LPS from each type of bacterium was extracted and purified by the hot phenolwater procedure of Westphal et al. (4). Human dental pulp cells (2 ml; 2.5 x 104 cells/ml) were seeded in culture dishes (35 mm diameter) in MEM supplemented with 10% FBS. After incubation for 24 h at 37"C in 5% CO2/95% air atmosphere, the cell monolayer was washed two times with calcium- and magnesium-free, phosphatebuffered saline solution. Thereafter, 2 ml of 10% FBS-MEM containing 1, 10, or 100 #g/ml of LPS was added to each of five dishes. Further incubation was done for 3 to 14 days; the DNA content, protein content, and alkaline phosphatase activity in cells were measured. The DNA content was measured by a modification of the method of Hinegardner (5); protein content, with a Bio-Rad Protein Assay kit (6); and alkaline phosphatase activity, by the procedure of Bessey et al. (7). All statistical evaluations were made with Student's t test.
130
Journal of Endodontics
Nakane et al.
RESULTS Production of DNA by pulpal cells was increased significantly by E. coil LPS at 10 ug/ml at 10 and 14 days (p < 0.01), and inhibited at 100 ~g/ml at all experimental periods (p < 0.01); increased by P. gingivalis LPS at 10 #g/ml at 3, 7, and 10 days (p < 0.05, p < 0.01) and inhibited by it at 100 ug/ml at 3 and 7 days (p < 0.01); decreased by P. endodontalis LPS at 100 #g/ml at 3 and 7 days (p < 0.01); and increased by F. nucleatum LPS at 10 jzg/ml at 3 and 7 days (p < 0.05) and decreased by it at 100 ug/ml at 3, 7, and 10 days (p < 0.01 ) (Fig. 1). E. coli LPS at 10 #g/ml inhibited pulp cell protein production significantly at 3 and 7 days (p < 0.01) and at 100 ug/ ml, at all experimental periods (p < 0.01); P. gingivalis LPS at 100 t~g/ml blocked it at 3, 7, and 10 days (p < 0.01); P. endodontalis LPS at 100 #g/ml, at all experimental periods (p < 0.01); and F. nucleatum LPS at 100 #g/ml, at 3, 7, and 10 days (p < 0.01) (Fig. 2). Alkaline phosphatase activity was unaffected by any LPS at any concentration throughout the experimental period (Fig. 3).
when the pulp is inflamed. However, there was no effect of any LPS on the differentiation state of human dental pulp cells. Schein and Schilder (11), Dahlen and Bergenholtz (12), and Horiba et al. (13) reported that the average value of the LPS content detected in the infected root canal ranged from 1 to 100 ~g/ml. Therefore the concentration of 1 to 100 ug/ ml of LPS was used in this study. It is known that the LPS has a cytotoxic effect on various cells, such as lymphocytes, human periodontal ligament-derived fibroblasts, and others, and stimulates them to produce cytokines like interleukin-l, interleukin-6, tumor necrosis factors, and so forth (14-16). These cytokines are mediators of inflammation. It would be very worthwhile to investigate the relationship between LPS and the release of cytokines from human dental pulp cells. Drs. Nakane, Yoshida, Nakata, Horiba, and Nakamura are members of the Department of Endodontics, School of Dentistry, Aichi Gakuin University, Nagoya, Japan. Address requests for reprints to Dr. Akinobu Nakane, Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya 464, Japan.
References
DISCUSSION Several reports have investigated a correlation between periapical disease and LPS content. Yamasaki et al. (8) reported a positive correlation between the development of periapical lesions and an increase in LPS content. However, few investigations have shown a correlation between the breakdown of pulp matrix and LPS content. Pinero et al. (2) reported that, when the uptake of [3H]thymidine by bovine pulp fibroblast cultures was measured in the cell fraction at 6, 24, and 48 h, there was an increase over the control when the cells were treated with 5 ug/ml of LPS purified from E. coil, no change with 125 ~g/ml, and an inhibitory effect with 625 ~g/ml. Isomura (9) reported that the LPS from F. nucleaturn showed cytotoxic effects on HeLa cells with an LDso concentration of 489 #g/ml. Also, Sugizaki (10) calculated the concentration of LPS for 50% inhibition of the control to be 280 t~g/ml. Our results indicate that production of DNA in human dental pulp cells was inhibited significantly at 100 ~g/ml LPS purified from E. coli at all experimental periods, thus differing from the findings of Pinero et al. (2). With respect to the sensitivity of cells to LPS, differences in the origin of the cells as well as differences in culture conditions may be important factors. Also, the discrepancy could be caused by the difference in LPS preparation. Production of protein in human dental pulp cells was inhibited at 10 ttg/ml of LPS from E. coil and was inhibited slightly at I0 #g/ml of LPS from P. gingivalis, P. endodontalis, and F. nucleatum. Our results showed that 10 ~g/ml of E. coli LPS provoked an increase in the production of DNA and a decrease in that of protein. The reason for this finding may be caused by a difference between the molecular structure of E. coli LPS and that of others. The alkaline phosphatase activity was measured to evaluate the effect of LPS on the differentiation ability of the cells. Large amounts of alkaline phosphatase are found in pulpal odontoblasts when the cells are active in calcification and
1. Hashioka K, Yamasaki M, Nakane A, Hodbe N, Nakamura H. The relationship between clinical symptoms and anaerobic bacteria from infected root canals. J Endodon 1992;18:558-61. 2. Pinero G, Kiatpongsah S, Hutchins MO, Hoover J. The effect of endotoxin on the synthesis of connective tissue matrix components by pulp fibroblasts in vitro. J Endodon 1983;9:2-7. 3. Hodba N, Maekawa Y, Abe Y, Ito M, Matsumoto T, Nakamura H. Correlations between endotoxin and clinical symptoms or radiolucent areas in infected root canals. Oral Surg 1991 ;71:492-5. 4. Westphal O, Jann K. Extraction with phenol-water and further applications of the procedure. Methods Carbohydrate Chem 1965;5:83-91. 5. Hinegardner RT. An improved fluorometric assay for DNA. Anal Biochem 1971 ;39:197-201. 6. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54. 7. Bessey OA, Lowry OH, Brock MJ. A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J Biol Chem 1946; 164:321-9. 8. Yamasaki M, Nakane A, Kumazawa M, Hashioka K, Hodbe N, Nakamura H. Endotoxin and gram-negative bacteria in the rat pedapical lesions. J Endodon 1992;18:501-4. 9. Isomura K. Studies on bacterial endotoxin from Fusobacterium nucleaturn--especially the role of endotoxin in periodontal diseases. J Jpn So(: Perodont 1985;27:328-51 (in Japanese). 10. Sugizaki K. Metabolic alteration of Gin-1 flbroblast by lipopolysaccharide from pedodontopathic bacteda. J Japan Soc Periodont 1986;28:468-83 (in Japanese). 11. Schein B, Schilder H. Endotoxin content in endodontically involved teeth. J Endodon 1975;1:19-21. 12. Dahlen G, Bergenholtz G. Eodotoxic activity in teeth with necrotic pulps. J Dent Res 1980;59:1033-40. 13. Horiba N, Maekawa Y, Matsumoto T, Nakamura H. A study of the distdbution of endotoxin in the dentinal wall of infected root canals. J Endodon 1990; 16:331-4. 14. Hameda S, Takada H, Ogawa T, Fujiwara T, Mihara J. Upopolysaccharides of oral anaerobes associated with chronic inflammation: chemical and immunomodulating properties. Intern Rev Immuno11990;6:247-61. 15. Takada T, Mihara J, Modsaki I, Hamada S. Induction of intedeukin-1 and -6 in human gingival fibroblast cultures stimulated with Bacteroides [ipopolysaccharides. Infect Immun 1991 ;59:295-301. 16. Yamazaki K, Ikarashi F, Aoyagi T, Takahashi K, Nakajima T, Hara K, Seymour GJ. Direct and indirect effects of Porphyromonas gingivalis lipopotysaccharide on intedeukin-6 production by human gingival fibroblasts. Oral Microbio11992;7:218-24.