Archs oral Bid. Vol. 37, No. 2, pp. 105-109, Printed in Great Britain. All rights reserved
1992
0003-9969/92 SS.00 + 0.00 Copyright 0 1992 Pergamon Press plc
EFFECTS OF MATERNAL CAFFEINE INTAKE DURING LACTATION ON MOLAR ENAMEL SURFACES IN NEW-BORN RATS K. HASHIMOTO,’ F. JOSEPHJR,~ A. U. FALSTER,~ W. B. SIMMONS’and T. NAKAMOTO’S* Departments of ‘Physiology and ‘Pediatrics, Laboratory of Perinatal Nutrition and Metabolism, Louisiana State University Medical Center, New Orleans, LA 70119 and rDepartment of Geology and Geophysics, University of New Orleans, Lakefront, New Orleans, LA 70148, U.S.A. (Accepted
16 August 1991)
Summary-:Dams
were fed normal laboratory chow until delivery. At birth, the litters were combined, and eight pups were randomly assigned to each dam. Dams with the recombined litters were divided into two groups. Dams of group 1 were fed a 20% protein diet as a control; dams of group 2 were fed a 20% protein diet supplemented with caffeine (2 mg/lOO g of the dam’s weight). On day 22, the dams of group 2 were anaesthetized with ether. They were injected with 2iu of oxytocin in order to collect milk. Blood was collected from pups and dams to determine its caffeine concentration, The first and second molars were removed frc’m each pup’s mandible and maxilla. Radiographs were taken of 10 randomly selected first or second molars from each group. Four randomly selected molars from each litter were placed in a specially des,igned chamber and bathed with a constant flow of acid solution to determine the amount of mineral dissolved from the enamel surfaces. The remaining non-acid exposed molars were pulverized in freezer mills. A small portion of this powder was then analyzed for the total amount of minerals. No differences were found in the radiographic density of enamel between the groups. The amount of dissolved calcium, phosphorus and magnesium from enamel surfaces in the caffeine group was consistently greater than that of the non-caffeine group in the first molars, whereas, in the second molars, there was no difference between the groups. The concentration of calcium, phosphorus and zinc in the first molars in the caffeine group was less than that in the non-caffeine group, whereas there was no difference between the groups in the second molars. Thus the caffeine intake of suckling pups in the early growing period affected the enamel surface of the first molar. Key words: caffeine, enamel surface, newborn rats.
INTRODUCTION Caffeine (1,3,7-trimethylxanthine) is one of the substances most frequently consumed in our daily life. It is contained in beverages, such as coffee, tea, cola and other carbonated soft drinks, and in over-the-counter medications (Graham, 1978). Caffeine diffuses easily in breast milk in humans (Aldridge, Aranda and Neims, 1975) and rats (Aeschbacher et al., 1980). It has been reported that, in recent years, 52% of women with more than 12 years of schooling are choosing to breast feed their infants (National Cancer Health Statistics, 1980). Furthermore, the increasing consumption of soft drinks means that people are increasing their caffeine intake. On a mg/kg; of body weight basis those in the l-5 yr age range have the highest caffeine intake in the United States (Abbott, 1986). Therefore, lactating women who consume caffeine could affect their offspring, who are rapidly growing, and a child who drinks soft drinks may also consume caffeine during periods of rapid growth. We have shown that maternal dietary caffeine intake during lactation alters the composition of the developing tooth in new-born rats (Nakamoto, Shaye and Mallek, 1985). The calcium content of the first *To whom all correspondence
should be addressed
molar in the caffeine-supplemented group was less than that in the non-caffeine group under conditions in which animals received normal nourishment. This change in the mineral content of the developing tooth may come from enamel, dentine or both. Impairment of the enamel surface of the developing tooth as a result of maternal caffeine intake and/or direct consumption of soft drinks could potentially result in a caries-prone tooth. Therefore, we have now sought to determine if caffeine intake during early growth can affect the enamel surface of the developing tooth in the new-born rat. MATERIALSAND METHODS Timed-pregnant rats, obtained from a commercial breeder, (Holtzman Co., Madison, WI) were used. At term, litters delivered within an 8 h period were combined and designated as day 1; eight pups were randomly assigned to each dam. Then, the dams with the recombined litters were divided into two groups: dams of group 1 (n = 6) were fed a 20% protein diet; those of group 2 (n = 7) were fed a 20% protein diet supplemented with caffeine. The composition of the diet has already been described (Quinby and Nakamoto, 1984). The caffeine supplementation was 2 mg/lOOg of the dam’s body weight. Because the dam’s body weight falls slightly from the time of 105
K. HASHIMOTOet
106
delivery, maternal caffeine supplementation was adjusted four times, at days 1, 6, 13 and 20, during the experiment. The technical details of caffeine supplementation have already been reported (Nakamoto and Shaye, 1986). On day 22, pups from each group were killed by decapitation. Blood was collected in heparinized tubes and centrifuged to obtain plasma. The four first and four second molars of each pup were removed, producing 32 first and 32 second molars per litter. As we had six litters in each group, we collected 192 first and 192 second molars in each group. First and second molars in each litter were combined and stored frozen at -40°C until further analysis. Dams were anaesthetized with ether and injected intraperitoneally with 2 iu of oxytocin (Sigma Chemical Co., St Louis, MO) (Keen et al., 1981). Milk was then collected in plastic tubes. Then the maternal blood was collected by cardiac puncture into heparinized tubes, centrifuged to obtain plasma, and stored at -40°C until further analysis. One or two first and second molars from each group in each litter were selected at random, up to a total of 10 of each tooth, and were dried at 110°C overnight. Radiographs were then taken of the teeth for density study under the following conditions. The distance between tooth and film (Kodak, ultraspeed dental film-occlusal type) was 7 in.; the distance between the focal spot and the teeth 20 in. Exposures were made using 90 kVp, 10 mA and 21 impulses. The exposed films were processed as follows. The automatic processor was the AT-2000 (Air Techniques, Inc., Hicksville, NY). Processing time was 5.5 min at 28°C. Kodak RP X-O matt developer and fixer solutions were used. The enamel portion of the films was scanned with a microphotometer (Sakura PDS 15, Tokyo, Japan). Scanning was horizontal from the most prominent point of the proximal surface to the point on the opposite side of the distal surface. The most calcified point of the lowest density was measured and recorded (Fig. 1).
al.
Four randomly selected first or second molars (each from a different litter) were mounted with a sticky wax on a small plastic block in order to study the acid solubility on the enamel surface, as described by Aponte-Merced and Navia (1980). The mounted teeth were then cleaned with deionized water and a brush attached to a dental engine. The exposed dentine on the occlusal cusp surfaces was covered with nail-varnish (Aponte-Merced and Navia, 1980). A specially designed, sealed chamber with openings on its opposite sides allowed inflow and outflow of the acid solution. The opposite side of the tube inflow to the chamber was attached to a syringe pump (Model 255-l; Sage Instrument Co., White Plains, NY). The teeth inside of the chamber were bathed at a constant flow rate with a 20 mM solution of acetic acid (pH 4.0, adjusted with 0.1 mM NaOH). A 20 ml disposable plastic syringe was filled with an acid solution at a constant flow rate of 1 ml/5 min. The effluent flow was collected from the other end. The teeth were exposed to acid dissolution for 80 min, and four fractional collections were made at 20-min intervals. The effluent flow (4 ml/20 min) was collected in disposable plastic tubes. The same procedures were repeated seven more times in each group, using a different group of molars. Chemical analyses of calcium and magnesium in the collected samples were by atomic absorption spectrophotometry (Model 280, Fisher Scientific Co., Fair Lawn, NJ). The phosphorus concentration was measured spectrophotometrically (Chen, Toribara and Warner, 1956). Twenty-eight randomly selected, non-acid exposed teeth from different litters were dried at 110°C overnight and weighed. These samples were powdered in a freezer mill (Spex Industries, Inc., Metuchen, NJ) for 3 min. A tiny portion of the powdered samples was weighed and placed in a porcelain crucible. This was then ashed at 600°C overnight and dissolved in 0.2-0.3 ml of concentrated HCl and diluted with double-distilled deionized water. Calcium, phosphorus and magnesium concentrations were measured as described previously. Zinc concentration was determined by atomic absorption spectrophotometry. The caffeine concentrations of milk and plasma of pups and dams were measured as described by Nakamoto et al. (1988). Statistical analyses used the Student r-test and linear regression analysis with correlation, with 5% considered significant. RESULTS
I
A
I
Fig. 1. Upper: diagram of the tooth as seen in the radiograph and the direction of scanning on enamel. Lower: typical scanned line on the chart. Point X indicates the lowest density of enamel.
One dam in the caffeine group ate all of the litter and was eliminated from the study. Mean body-weight gains over the experimental period are shown in Fig. 2; there was no significant difference on any day between the control and caffeine groups (p > 0.05). Radiographic enamel densities measured from scanned paper of the first and second molars in the control and caffeine groups were as follows (mean + SEM): control (first, 1.09 f 0.01; second, 1.12 k 0.08) caffeine (first, 1.10 + 0.0 1; second, 1.13 + 0.06). There was no significant difference in radiographic enamel density in either the first or second molars between the groups (p > 0.05).
Enamel surface after caffeine 60
r 0
107
Control
Iz] Cuffeine 60
t
Total amount of P
dissolved
(la1
molar)
60-f
6
2
14
IO
16
22
Days
Fig. 2. Changes
in body weight of pups experimental period.
over the
,i The mean amount of enamel minerals dissolved from the first and second molars during each time interval are shown in Table 1. However, Fig. 3 shows the mean total calcium, phosphorus and magnesium dissolved over 80 min of acid dissolution as pg per four of first molars; each individual value of the respective minerals dissolved at each 20-min interval was added cumullatively. The amount of calcium, phosphorus and magnesium dissolved from the enamel surface of the first molars in the caffeine group was significantly higher during each time interval than those of the controls (p < 0.05), whereas in the second molars, those variables showed no significant difference between the groups (p > 0.05) (Table I). The cumulative amount of calcium, phosphorus and magnesium djssolved from the enamel at 20, 40, 60 and 80 min was higher for the caffeine group than for the controls 01 < 0.05) (Fig. 3). The total amount of calcium, phosphorus, magnesium and zinc concentration of the first molars in the caffeine group was significantly less than that
.+/, 0
(
2.0 Time
3.0 -f 2.0 2.0 CI
a.
1.5 1.0
4.0
Total amount of Mg
dissolved
(1st
L-&!& r.l.OO
0
ZO
Time
40
0
cO”tml
l
CaeaiIy
00
Fig. 3. Means of total calcium, phosphorus and magnesium dissolved over 80 min of acid exposure as pg per four first molars. Each individual value from Table 1 was added cumulatively.
of the controls (p < 0.05), whereas in the second molars there was no significant difference between the groups (p > 0.05) (Table 2).
Second molar
First molar Caffeine
Control
5.23 + 0.33 2.67 + 0.36 0.20 f 0.02
20 8.63 k 1.07* 5.66 Jtr 1.07; 0.44 * 0.07.
min
Ca P Mi:
10.74 + 1.17 7.34 * 0.77 0.39 + 0.06
40 16.83 5 1.10. 12.49 k 1.40’ 0.72 + 0.06’
min
Ca P M:g Ca P M::
12.36 k 1.22 8.03 + 0.78 0.43 f 0.04
Ca P
13.68 & 1.30 9.39 + 0.91 0.42 + 0.04
ME
80
(mlnutrr)
Table 1. Mean amount of minerals dissolved from first or second molars during each individual time interval (bg/four first or second molars)
Control
molar)
Ii1 .oo
0.0 0.0
0-0
10
(minutes)
Caffeine
7.48 k 1.29 4.09 + 0.98 0.28 k 0.04
7.36 f 0.86 4.41 & 0.89 0.31 + 0.04
13.59 & 1.15 10.03 * 1.45 0.45 + 0.04
13.63 + 0.92 8.68 * 0.95 0.55 f 0.05
60 min 18.19 f 1.12; 14.68 f 14.72 + 1.98* 10.47 * 0.74 + 0.06* 0.49 + 80 min 19.64 &-1.19* 15.21 k 18.12 k 2.62* 11.05 + 0.71 + 0.05* 0.43 *
1.15 1.15 0.03
14.85 + 1.19 10.42 k 0.97 0.55 + 0.05
1.22 1.10 0.05
15.94 f 0.91 12.88 f 1.80 0.52 f 0.05
Msean + SEM. Each value is an average of seven determinations. *Significantly different from the control at p < 0.05.
K. HAWMOTOet al.
108
Table 2. Various mineral concentrations (unit/g of pulverized tissue) of whole teeth after pulverization Control
Caffeine First Molars
Ca (w) P (mid Mg (mg) Zn old
288.8 + 141.0 + 2.44 7 127.3 k
6.8 5.3 0.10 7.0
Ca (mg) P (mg) Mg (mg) Zn (fl g)
286.5 + 121.3 + 2.23 + 93.8 k
9.2 6.0 0.10 3.4
270.5 k 2.1* 121.8 + 2.9* 2.15 *0.05* 100.0 * 4.0*
Second Molars
281.0 f 123.0 k 2.10 + 106.0 +
6.7 8.4 0.14 6.4
Mean f SEM. Each value is an average of four determinations. *Significantly different from the control at p < 0.05.
Caffeine concentration of milk bg/ml) was 0.679 +_0.211 (mean f SEM). Plasma caffeine concentration @g/ml) of the dams and pups was 0.825 + 0.319 and 0.211 f 0.042, respectively. DISCUSSION
Caffeine was not used here in large pharmacological dosages. Assuming the caffeine content of a cup of coffee to be 100 mg, the amount of caffeine consumption in our study is equivalent to a daily intake of 10 cups of coffee for a 50 kg woman (Gilbert and Pistey, 1973). Caffeine intake for the 99th percentile of 18 y or older is reported to be nearly 7 cups of coffee per day (Graham, 1978). Therefore, it is possible that those women who are breast feeding could be consuming the equivalent of 10 cups of coffee a day. When the conversion is based on the metabolic body weight (kg0.75)(Kleiber, 1961), the caffeine intake of the same person becomes slightly more than that contained in two cups of coffee. Pups of both groups grew with the same rapidity, suggesting that the amount of caffeine present did not exert any toxic effects on them. In an earlier study of caffeine’s effects on the developing teeth of new-born rats (Nakamoto et al., 1985), the teeth of the suckling offspring whose dams were fed a diet supplemented with caffeine were affected by caffeine in the milk. We now specifically examined the enamel surface. Any changes in chemical composition of the enamel due to caffeine intake in the early growing period may alter susceptibility to dental caries. Nutritional deficiency, such as protein deficiency, is reported to affect mineralization of the enamel and may lead to an increased susceptibility to dental caries (Navia et al., 1970). We took radiographs of randomly selected first and second molars to determine whether these could show any clinical changes at the enamel surface. Radiographic film showed no difference in density between the groups. Thus, one can reasonably conclude that clinical observation could not detect any changes in the enamel of the offspring as a result of early caffeine intake. Decreased acid resistance to the enamel surface is reported to be an indication of caries susceptibility (Aponte-Merced and Navia, 1980). We found greater dissolution of calcium, phosphorus and magnesium content in the first molars of the caffeine group than
in the non-caffeine controls. This suggests that the teeth from the caffeine group could be less resistant to dental caries than those of the non-caffeine controls in a given circumstance. The percentage differences of acid-dissolved calcium, phosphorus and magnesium between the groups in the initial 20 min were greater than those at the end of 80 min. As enamel dissolved further from 20 to 80 min, the percentage difference between the groups decreased. The outer surface of the enamel was apparently affected more in its chemical composition than the inner layer. Non-caffeine controls showed lower acid solubility than the caffeine group throughout the 80min of the experiment. The acid solubility of the second molars was different; there was no significant difference in the amount of acid-dissolved minerals between the group over any interval. The dissolved calcium, phosphorus and magnesium contents of the first molars in the non-caffeine control were consistently less than those of the second molars of the non-caffeine control during each time interval. The first appearance in the mouth of the first and second molars is reported to be on day 19 and 22, respectively (&hour and Massler, 1949). If posteruptive mineral deposition and maturation are critical and accomplished by mineral present in the oral fluids (Aponte-Merced and Navia, 1980), then differences in posteruptive mineralization between first molars already erupted and second molars still in the eruptive stage could be one of the reasons for the lowered solubility of the tist molars of the non-caffeine group. The dissolved calcium, phosphorus and magnesium contents of the first molars in the caffeine group were consistently greater than those of the second molars of the caffeine group during each time interval. It is conceivable that postnatal caffeine intake by the pups could have exerted effects on the matrix formation and/or mineralization stages of the enamel, thus changing its composition. However, because of the different chronological stages of amelogenesis between first and second molars (Schour and Massler, 1949), caffeine intake did not play a critical role in the formation of the second molars. This argument can be justified by the fact that the dissolved minerals in the second molars were practically identical between non-caffeine control and caffeine groups. The total amount of various mineral concentrations of the first molars in the caffeine group was consistently lower than that of the non-caffeine group. Recent studies have shown that dietary caffeine decreases the zinc levels of the fetal bones (Nakamoto, Grant and Yazdani, 1989a), brain (Nakamoto, Hartman and Joseph, 1989b), and heart (Rossowska et al., 1990). Suckling pups whose dams were fed a zinc-deficient diet have been shown to have an increased incidence in dental caries (Brown et al., 1979). There was no significant difference between groups in the various mineral concentrations in the second molars. Caffeine intake, under our experimental conditions, had a minor effect, indicating that the role of caffeine in the development of second molars during this time is not critical. The caffeine concentrations in milk and plasma of the dams and pups confirm that they had been
Enamel surface after caffeine
exposed to caffeine during early growth. Extrapolation of findings from the rat to man should be made with extreme caution. However, breast feeding is on the increase (National Cancer Health Statistics, 1980) by mothers who may use caffeine routinely, and children’s consumption of soft drinks is high (Abbott, 1986). These factors could cause fundamental changes in the chemical composition of the enamel. Our data are the first evidence in animal models that maternal caffeine intake could impair the enamel of the developing teeth in the offspring, possibly increasing their susceptibility to dental caries. Acknowledgements--We wish to express sincere appreciation for the help provided by MS G. Young and Dr M. J. Rossowska, and MI M. Higgins, Editorial Consultant. Dr K. Hashimoto was supported by a fellowship grant from Nihon University, Tokyo, Japan. This work was supported in part by grant BRSG SO7 RR05704-15 DRR, NIH.
REFERENCES Abbott P. J. (1986) Caffeine: a toxicological overview. Med. J. Aust. 14!i, 518-521. Aeschbacher H. U., Milton H., Pott A. and Wurzner H. P. (1980) Effect of caffeine on rat offspring from treated dams. Toxic. Let!. 7, 71-77. Aldridge A., Aranda J. V. and Neims A. M. (1975) Caffeine metabolism in the newborn. Clin. Pharmac. Ther. 25, 977-98 1. Aponte-Merced L. and Navia J. M. (1980) Pre-eruptive protein-energy malnutrition and acid solubility of rat molar enamel surfaces. Archs oral Biol. 25, 701-705. Brown E. D., Calhoun N. R., Larson R. H. and Smith J. C. Jr (1979) An effect of zinc deficiency on dental caries. I;ife SC; 24, ‘2093-2098. Chen P. S.. Toribara T. Y. and Warner H. (1956) Microdetermination of phosphorus. Analyt. Chem. 28; 175661758. Gilbert E. F. and Pistey W. R. (1973) Effect on the offspring of repeated cafi:ine administration to pregnant rats. J. Reprod. Fert. 34, 494-499.
109
Graham D. M. (1978)Caffeine-its identity, dietary sources, intake and biological effects. Nutr. Rev. 36, 97-102. Keen C. L., Lonnerdal B., Clegg M. and Hurley L. S. (1981) Developmental changes in composition of rat milk: trace elements, minerals, protein, carbohydrate and fat. J. Nun. 111, 226-230. Kleiber M. (1961) Body size and metabolic rate. In The Fire of Life, an Introduction to Animal Energetics, pp. 177-216. Wiley, New York. Nakamoto T. and Shaye R. (1986) Protein-energy malnutrition in rats during pregnancy modifies the effects of caffeine on fetal bones. J. Nutr. 116, 633-640. Nakamoto T., Shaye R. and Mallek H. M. (1985) Effects of maternal caffeine intake on the growth of tooth germs in protein-energy malnourished neonates. Archs oral Biol. 30, 105-109. Nakamoto T., Joseph F. Jr, Yazdani M. and Hartman A. D. (1988) Effects of different levels of caffeine supplemented to the maternal diet on the brains of newborn rats and their dams. Toxic. Lett. 44, 167-175. Nakamoto T., Grant S. and Yazdani M. (1989a) The effects of maternal caffeine intake during pregnancy on mineral contents of fetal rat bone. Res. exp. Med. 189, 275-280. Nakamoto T., Hartman A. D. and Joseph F. Jr (1989b) Interaction between caffeine intake and nutritional status on growing brains in newborn rats. Ann. Nutr. Metab. 33, 92-99. National Cancer Health Statistics (1980) Advance data: trends in breast feeding. Vital and Health Statistics. DHEW No. 80-1250. Navia J. M., DiOrio C. P., Menaker L. and Miller S. A. (1970) Effect of undernutrition during the perinatal period on caries development in the rat. J. dent. Res. 49, 1091-1098. Quinby G. E. and Nakamoto T. (1984) Theophylline effects on cellular response in protein-energy malnourished neonatal rat brain. Pediutr. Res. 18, 546-549. Rossowska M. J., Dinh C., Gottschalk S. B., Yaxdani M., Sutton F. S. III and Nakamoto T. (1990) Interaction between caffeine intake and heart zinc concentration in the rats. Br. J. Nutr. 64, 561-567. Schour I. and Massler M. (1949) The teeth. In The Rat in Laboratory Investigation (Eds Farris E. J. and Griffith J. Q.), 2nd edn, pp. 106. J. B. Lippencott Comp., Philadelphia, PA.
1992
0003-9969/92 SS.00 + 0.00 Copyright 0 1992 Pergamon Press plc
EFFECTS OF MATERNAL CAFFEINE INTAKE DURING LACTATION ON MOLAR ENAMEL SURFACES IN NEW-BORN RATS K. HASHIMOTO,’ F. JOSEPHJR,~ A. U. FALSTER,~ W. B. SIMMONS’and T. NAKAMOTO’S* Departments of ‘Physiology and ‘Pediatrics, Laboratory of Perinatal Nutrition and Metabolism, Louisiana State University Medical Center, New Orleans, LA 70119 and rDepartment of Geology and Geophysics, University of New Orleans, Lakefront, New Orleans, LA 70148, U.S.A. (Accepted
16 August 1991)
Summary-:Dams
were fed normal laboratory chow until delivery. At birth, the litters were combined, and eight pups were randomly assigned to each dam. Dams with the recombined litters were divided into two groups. Dams of group 1 were fed a 20% protein diet as a control; dams of group 2 were fed a 20% protein diet supplemented with caffeine (2 mg/lOO g of the dam’s weight). On day 22, the dams of group 2 were anaesthetized with ether. They were injected with 2iu of oxytocin in order to collect milk. Blood was collected from pups and dams to determine its caffeine concentration, The first and second molars were removed frc’m each pup’s mandible and maxilla. Radiographs were taken of 10 randomly selected first or second molars from each group. Four randomly selected molars from each litter were placed in a specially des,igned chamber and bathed with a constant flow of acid solution to determine the amount of mineral dissolved from the enamel surfaces. The remaining non-acid exposed molars were pulverized in freezer mills. A small portion of this powder was then analyzed for the total amount of minerals. No differences were found in the radiographic density of enamel between the groups. The amount of dissolved calcium, phosphorus and magnesium from enamel surfaces in the caffeine group was consistently greater than that of the non-caffeine group in the first molars, whereas, in the second molars, there was no difference between the groups. The concentration of calcium, phosphorus and zinc in the first molars in the caffeine group was less than that in the non-caffeine group, whereas there was no difference between the groups in the second molars. Thus the caffeine intake of suckling pups in the early growing period affected the enamel surface of the first molar. Key words: caffeine, enamel surface, newborn rats.
INTRODUCTION Caffeine (1,3,7-trimethylxanthine) is one of the substances most frequently consumed in our daily life. It is contained in beverages, such as coffee, tea, cola and other carbonated soft drinks, and in over-the-counter medications (Graham, 1978). Caffeine diffuses easily in breast milk in humans (Aldridge, Aranda and Neims, 1975) and rats (Aeschbacher et al., 1980). It has been reported that, in recent years, 52% of women with more than 12 years of schooling are choosing to breast feed their infants (National Cancer Health Statistics, 1980). Furthermore, the increasing consumption of soft drinks means that people are increasing their caffeine intake. On a mg/kg; of body weight basis those in the l-5 yr age range have the highest caffeine intake in the United States (Abbott, 1986). Therefore, lactating women who consume caffeine could affect their offspring, who are rapidly growing, and a child who drinks soft drinks may also consume caffeine during periods of rapid growth. We have shown that maternal dietary caffeine intake during lactation alters the composition of the developing tooth in new-born rats (Nakamoto, Shaye and Mallek, 1985). The calcium content of the first *To whom all correspondence
should be addressed
molar in the caffeine-supplemented group was less than that in the non-caffeine group under conditions in which animals received normal nourishment. This change in the mineral content of the developing tooth may come from enamel, dentine or both. Impairment of the enamel surface of the developing tooth as a result of maternal caffeine intake and/or direct consumption of soft drinks could potentially result in a caries-prone tooth. Therefore, we have now sought to determine if caffeine intake during early growth can affect the enamel surface of the developing tooth in the new-born rat. MATERIALSAND METHODS Timed-pregnant rats, obtained from a commercial breeder, (Holtzman Co., Madison, WI) were used. At term, litters delivered within an 8 h period were combined and designated as day 1; eight pups were randomly assigned to each dam. Then, the dams with the recombined litters were divided into two groups: dams of group 1 (n = 6) were fed a 20% protein diet; those of group 2 (n = 7) were fed a 20% protein diet supplemented with caffeine. The composition of the diet has already been described (Quinby and Nakamoto, 1984). The caffeine supplementation was 2 mg/lOOg of the dam’s body weight. Because the dam’s body weight falls slightly from the time of 105
K. HASHIMOTOet
106
delivery, maternal caffeine supplementation was adjusted four times, at days 1, 6, 13 and 20, during the experiment. The technical details of caffeine supplementation have already been reported (Nakamoto and Shaye, 1986). On day 22, pups from each group were killed by decapitation. Blood was collected in heparinized tubes and centrifuged to obtain plasma. The four first and four second molars of each pup were removed, producing 32 first and 32 second molars per litter. As we had six litters in each group, we collected 192 first and 192 second molars in each group. First and second molars in each litter were combined and stored frozen at -40°C until further analysis. Dams were anaesthetized with ether and injected intraperitoneally with 2 iu of oxytocin (Sigma Chemical Co., St Louis, MO) (Keen et al., 1981). Milk was then collected in plastic tubes. Then the maternal blood was collected by cardiac puncture into heparinized tubes, centrifuged to obtain plasma, and stored at -40°C until further analysis. One or two first and second molars from each group in each litter were selected at random, up to a total of 10 of each tooth, and were dried at 110°C overnight. Radiographs were then taken of the teeth for density study under the following conditions. The distance between tooth and film (Kodak, ultraspeed dental film-occlusal type) was 7 in.; the distance between the focal spot and the teeth 20 in. Exposures were made using 90 kVp, 10 mA and 21 impulses. The exposed films were processed as follows. The automatic processor was the AT-2000 (Air Techniques, Inc., Hicksville, NY). Processing time was 5.5 min at 28°C. Kodak RP X-O matt developer and fixer solutions were used. The enamel portion of the films was scanned with a microphotometer (Sakura PDS 15, Tokyo, Japan). Scanning was horizontal from the most prominent point of the proximal surface to the point on the opposite side of the distal surface. The most calcified point of the lowest density was measured and recorded (Fig. 1).
al.
Four randomly selected first or second molars (each from a different litter) were mounted with a sticky wax on a small plastic block in order to study the acid solubility on the enamel surface, as described by Aponte-Merced and Navia (1980). The mounted teeth were then cleaned with deionized water and a brush attached to a dental engine. The exposed dentine on the occlusal cusp surfaces was covered with nail-varnish (Aponte-Merced and Navia, 1980). A specially designed, sealed chamber with openings on its opposite sides allowed inflow and outflow of the acid solution. The opposite side of the tube inflow to the chamber was attached to a syringe pump (Model 255-l; Sage Instrument Co., White Plains, NY). The teeth inside of the chamber were bathed at a constant flow rate with a 20 mM solution of acetic acid (pH 4.0, adjusted with 0.1 mM NaOH). A 20 ml disposable plastic syringe was filled with an acid solution at a constant flow rate of 1 ml/5 min. The effluent flow was collected from the other end. The teeth were exposed to acid dissolution for 80 min, and four fractional collections were made at 20-min intervals. The effluent flow (4 ml/20 min) was collected in disposable plastic tubes. The same procedures were repeated seven more times in each group, using a different group of molars. Chemical analyses of calcium and magnesium in the collected samples were by atomic absorption spectrophotometry (Model 280, Fisher Scientific Co., Fair Lawn, NJ). The phosphorus concentration was measured spectrophotometrically (Chen, Toribara and Warner, 1956). Twenty-eight randomly selected, non-acid exposed teeth from different litters were dried at 110°C overnight and weighed. These samples were powdered in a freezer mill (Spex Industries, Inc., Metuchen, NJ) for 3 min. A tiny portion of the powdered samples was weighed and placed in a porcelain crucible. This was then ashed at 600°C overnight and dissolved in 0.2-0.3 ml of concentrated HCl and diluted with double-distilled deionized water. Calcium, phosphorus and magnesium concentrations were measured as described previously. Zinc concentration was determined by atomic absorption spectrophotometry. The caffeine concentrations of milk and plasma of pups and dams were measured as described by Nakamoto et al. (1988). Statistical analyses used the Student r-test and linear regression analysis with correlation, with 5% considered significant. RESULTS
I
A
I
Fig. 1. Upper: diagram of the tooth as seen in the radiograph and the direction of scanning on enamel. Lower: typical scanned line on the chart. Point X indicates the lowest density of enamel.
One dam in the caffeine group ate all of the litter and was eliminated from the study. Mean body-weight gains over the experimental period are shown in Fig. 2; there was no significant difference on any day between the control and caffeine groups (p > 0.05). Radiographic enamel densities measured from scanned paper of the first and second molars in the control and caffeine groups were as follows (mean + SEM): control (first, 1.09 f 0.01; second, 1.12 k 0.08) caffeine (first, 1.10 + 0.0 1; second, 1.13 + 0.06). There was no significant difference in radiographic enamel density in either the first or second molars between the groups (p > 0.05).
Enamel surface after caffeine 60
r 0
107
Control
Iz] Cuffeine 60
t
Total amount of P
dissolved
(la1
molar)
60-f
6
2
14
IO
16
22
Days
Fig. 2. Changes
in body weight of pups experimental period.
over the
,i The mean amount of enamel minerals dissolved from the first and second molars during each time interval are shown in Table 1. However, Fig. 3 shows the mean total calcium, phosphorus and magnesium dissolved over 80 min of acid dissolution as pg per four of first molars; each individual value of the respective minerals dissolved at each 20-min interval was added cumullatively. The amount of calcium, phosphorus and magnesium dissolved from the enamel surface of the first molars in the caffeine group was significantly higher during each time interval than those of the controls (p < 0.05), whereas in the second molars, those variables showed no significant difference between the groups (p > 0.05) (Table I). The cumulative amount of calcium, phosphorus and magnesium djssolved from the enamel at 20, 40, 60 and 80 min was higher for the caffeine group than for the controls 01 < 0.05) (Fig. 3). The total amount of calcium, phosphorus, magnesium and zinc concentration of the first molars in the caffeine group was significantly less than that
.+/, 0
(
2.0 Time
3.0 -f 2.0 2.0 CI
a.
1.5 1.0
4.0
Total amount of Mg
dissolved
(1st
L-&!& r.l.OO
0
ZO
Time
40
0
cO”tml
l
CaeaiIy
00
Fig. 3. Means of total calcium, phosphorus and magnesium dissolved over 80 min of acid exposure as pg per four first molars. Each individual value from Table 1 was added cumulatively.
of the controls (p < 0.05), whereas in the second molars there was no significant difference between the groups (p > 0.05) (Table 2).
Second molar
First molar Caffeine
Control
5.23 + 0.33 2.67 + 0.36 0.20 f 0.02
20 8.63 k 1.07* 5.66 Jtr 1.07; 0.44 * 0.07.
min
Ca P Mi:
10.74 + 1.17 7.34 * 0.77 0.39 + 0.06
40 16.83 5 1.10. 12.49 k 1.40’ 0.72 + 0.06’
min
Ca P M:g Ca P M::
12.36 k 1.22 8.03 + 0.78 0.43 f 0.04
Ca P
13.68 & 1.30 9.39 + 0.91 0.42 + 0.04
ME
80
(mlnutrr)
Table 1. Mean amount of minerals dissolved from first or second molars during each individual time interval (bg/four first or second molars)
Control
molar)
Ii1 .oo
0.0 0.0
0-0
10
(minutes)
Caffeine
7.48 k 1.29 4.09 + 0.98 0.28 k 0.04
7.36 f 0.86 4.41 & 0.89 0.31 + 0.04
13.59 & 1.15 10.03 * 1.45 0.45 + 0.04
13.63 + 0.92 8.68 * 0.95 0.55 f 0.05
60 min 18.19 f 1.12; 14.68 f 14.72 + 1.98* 10.47 * 0.74 + 0.06* 0.49 + 80 min 19.64 &-1.19* 15.21 k 18.12 k 2.62* 11.05 + 0.71 + 0.05* 0.43 *
1.15 1.15 0.03
14.85 + 1.19 10.42 k 0.97 0.55 + 0.05
1.22 1.10 0.05
15.94 f 0.91 12.88 f 1.80 0.52 f 0.05
Msean + SEM. Each value is an average of seven determinations. *Significantly different from the control at p < 0.05.
K. HAWMOTOet al.
108
Table 2. Various mineral concentrations (unit/g of pulverized tissue) of whole teeth after pulverization Control
Caffeine First Molars
Ca (w) P (mid Mg (mg) Zn old
288.8 + 141.0 + 2.44 7 127.3 k
6.8 5.3 0.10 7.0
Ca (mg) P (mg) Mg (mg) Zn (fl g)
286.5 + 121.3 + 2.23 + 93.8 k
9.2 6.0 0.10 3.4
270.5 k 2.1* 121.8 + 2.9* 2.15 *0.05* 100.0 * 4.0*
Second Molars
281.0 f 123.0 k 2.10 + 106.0 +
6.7 8.4 0.14 6.4
Mean f SEM. Each value is an average of four determinations. *Significantly different from the control at p < 0.05.
Caffeine concentration of milk bg/ml) was 0.679 +_0.211 (mean f SEM). Plasma caffeine concentration @g/ml) of the dams and pups was 0.825 + 0.319 and 0.211 f 0.042, respectively. DISCUSSION
Caffeine was not used here in large pharmacological dosages. Assuming the caffeine content of a cup of coffee to be 100 mg, the amount of caffeine consumption in our study is equivalent to a daily intake of 10 cups of coffee for a 50 kg woman (Gilbert and Pistey, 1973). Caffeine intake for the 99th percentile of 18 y or older is reported to be nearly 7 cups of coffee per day (Graham, 1978). Therefore, it is possible that those women who are breast feeding could be consuming the equivalent of 10 cups of coffee a day. When the conversion is based on the metabolic body weight (kg0.75)(Kleiber, 1961), the caffeine intake of the same person becomes slightly more than that contained in two cups of coffee. Pups of both groups grew with the same rapidity, suggesting that the amount of caffeine present did not exert any toxic effects on them. In an earlier study of caffeine’s effects on the developing teeth of new-born rats (Nakamoto et al., 1985), the teeth of the suckling offspring whose dams were fed a diet supplemented with caffeine were affected by caffeine in the milk. We now specifically examined the enamel surface. Any changes in chemical composition of the enamel due to caffeine intake in the early growing period may alter susceptibility to dental caries. Nutritional deficiency, such as protein deficiency, is reported to affect mineralization of the enamel and may lead to an increased susceptibility to dental caries (Navia et al., 1970). We took radiographs of randomly selected first and second molars to determine whether these could show any clinical changes at the enamel surface. Radiographic film showed no difference in density between the groups. Thus, one can reasonably conclude that clinical observation could not detect any changes in the enamel of the offspring as a result of early caffeine intake. Decreased acid resistance to the enamel surface is reported to be an indication of caries susceptibility (Aponte-Merced and Navia, 1980). We found greater dissolution of calcium, phosphorus and magnesium content in the first molars of the caffeine group than
in the non-caffeine controls. This suggests that the teeth from the caffeine group could be less resistant to dental caries than those of the non-caffeine controls in a given circumstance. The percentage differences of acid-dissolved calcium, phosphorus and magnesium between the groups in the initial 20 min were greater than those at the end of 80 min. As enamel dissolved further from 20 to 80 min, the percentage difference between the groups decreased. The outer surface of the enamel was apparently affected more in its chemical composition than the inner layer. Non-caffeine controls showed lower acid solubility than the caffeine group throughout the 80min of the experiment. The acid solubility of the second molars was different; there was no significant difference in the amount of acid-dissolved minerals between the group over any interval. The dissolved calcium, phosphorus and magnesium contents of the first molars in the non-caffeine control were consistently less than those of the second molars of the non-caffeine control during each time interval. The first appearance in the mouth of the first and second molars is reported to be on day 19 and 22, respectively (&hour and Massler, 1949). If posteruptive mineral deposition and maturation are critical and accomplished by mineral present in the oral fluids (Aponte-Merced and Navia, 1980), then differences in posteruptive mineralization between first molars already erupted and second molars still in the eruptive stage could be one of the reasons for the lowered solubility of the tist molars of the non-caffeine group. The dissolved calcium, phosphorus and magnesium contents of the first molars in the caffeine group were consistently greater than those of the second molars of the caffeine group during each time interval. It is conceivable that postnatal caffeine intake by the pups could have exerted effects on the matrix formation and/or mineralization stages of the enamel, thus changing its composition. However, because of the different chronological stages of amelogenesis between first and second molars (Schour and Massler, 1949), caffeine intake did not play a critical role in the formation of the second molars. This argument can be justified by the fact that the dissolved minerals in the second molars were practically identical between non-caffeine control and caffeine groups. The total amount of various mineral concentrations of the first molars in the caffeine group was consistently lower than that of the non-caffeine group. Recent studies have shown that dietary caffeine decreases the zinc levels of the fetal bones (Nakamoto, Grant and Yazdani, 1989a), brain (Nakamoto, Hartman and Joseph, 1989b), and heart (Rossowska et al., 1990). Suckling pups whose dams were fed a zinc-deficient diet have been shown to have an increased incidence in dental caries (Brown et al., 1979). There was no significant difference between groups in the various mineral concentrations in the second molars. Caffeine intake, under our experimental conditions, had a minor effect, indicating that the role of caffeine in the development of second molars during this time is not critical. The caffeine concentrations in milk and plasma of the dams and pups confirm that they had been
Enamel surface after caffeine
exposed to caffeine during early growth. Extrapolation of findings from the rat to man should be made with extreme caution. However, breast feeding is on the increase (National Cancer Health Statistics, 1980) by mothers who may use caffeine routinely, and children’s consumption of soft drinks is high (Abbott, 1986). These factors could cause fundamental changes in the chemical composition of the enamel. Our data are the first evidence in animal models that maternal caffeine intake could impair the enamel of the developing teeth in the offspring, possibly increasing their susceptibility to dental caries. Acknowledgements--We wish to express sincere appreciation for the help provided by MS G. Young and Dr M. J. Rossowska, and MI M. Higgins, Editorial Consultant. Dr K. Hashimoto was supported by a fellowship grant from Nihon University, Tokyo, Japan. This work was supported in part by grant BRSG SO7 RR05704-15 DRR, NIH.
REFERENCES Abbott P. J. (1986) Caffeine: a toxicological overview. Med. J. Aust. 14!i, 518-521. Aeschbacher H. U., Milton H., Pott A. and Wurzner H. P. (1980) Effect of caffeine on rat offspring from treated dams. Toxic. Let!. 7, 71-77. Aldridge A., Aranda J. V. and Neims A. M. (1975) Caffeine metabolism in the newborn. Clin. Pharmac. Ther. 25, 977-98 1. Aponte-Merced L. and Navia J. M. (1980) Pre-eruptive protein-energy malnutrition and acid solubility of rat molar enamel surfaces. Archs oral Biol. 25, 701-705. Brown E. D., Calhoun N. R., Larson R. H. and Smith J. C. Jr (1979) An effect of zinc deficiency on dental caries. I;ife SC; 24, ‘2093-2098. Chen P. S.. Toribara T. Y. and Warner H. (1956) Microdetermination of phosphorus. Analyt. Chem. 28; 175661758. Gilbert E. F. and Pistey W. R. (1973) Effect on the offspring of repeated cafi:ine administration to pregnant rats. J. Reprod. Fert. 34, 494-499.
109
Graham D. M. (1978)Caffeine-its identity, dietary sources, intake and biological effects. Nutr. Rev. 36, 97-102. Keen C. L., Lonnerdal B., Clegg M. and Hurley L. S. (1981) Developmental changes in composition of rat milk: trace elements, minerals, protein, carbohydrate and fat. J. Nun. 111, 226-230. Kleiber M. (1961) Body size and metabolic rate. In The Fire of Life, an Introduction to Animal Energetics, pp. 177-216. Wiley, New York. Nakamoto T. and Shaye R. (1986) Protein-energy malnutrition in rats during pregnancy modifies the effects of caffeine on fetal bones. J. Nutr. 116, 633-640. Nakamoto T., Shaye R. and Mallek H. M. (1985) Effects of maternal caffeine intake on the growth of tooth germs in protein-energy malnourished neonates. Archs oral Biol. 30, 105-109. Nakamoto T., Joseph F. Jr, Yazdani M. and Hartman A. D. (1988) Effects of different levels of caffeine supplemented to the maternal diet on the brains of newborn rats and their dams. Toxic. Lett. 44, 167-175. Nakamoto T., Grant S. and Yazdani M. (1989a) The effects of maternal caffeine intake during pregnancy on mineral contents of fetal rat bone. Res. exp. Med. 189, 275-280. Nakamoto T., Hartman A. D. and Joseph F. Jr (1989b) Interaction between caffeine intake and nutritional status on growing brains in newborn rats. Ann. Nutr. Metab. 33, 92-99. National Cancer Health Statistics (1980) Advance data: trends in breast feeding. Vital and Health Statistics. DHEW No. 80-1250. Navia J. M., DiOrio C. P., Menaker L. and Miller S. A. (1970) Effect of undernutrition during the perinatal period on caries development in the rat. J. dent. Res. 49, 1091-1098. Quinby G. E. and Nakamoto T. (1984) Theophylline effects on cellular response in protein-energy malnourished neonatal rat brain. Pediutr. Res. 18, 546-549. Rossowska M. J., Dinh C., Gottschalk S. B., Yaxdani M., Sutton F. S. III and Nakamoto T. (1990) Interaction between caffeine intake and heart zinc concentration in the rats. Br. J. Nutr. 64, 561-567. Schour I. and Massler M. (1949) The teeth. In The Rat in Laboratory Investigation (Eds Farris E. J. and Griffith J. Q.), 2nd edn, pp. 106. J. B. Lippencott Comp., Philadelphia, PA.
