Analysis of Lead in Circumpulpal Dentin of Deciduous Teeth* PHILIPPE GRANDJEAN, M.D., PHD.,T OLE N0RBY HANSEN, M.P S Y C H ., and KAREN LYNGBYE, D.D.M

Analysis of Lead in Circumpulpal Dentin of Deciduous Teeth* PHILIPPE GRANDJEAN, M.D., PHD.,t OLE N0RBY HANSEN, M.P s y c h ., and KAREN LYNGBYE, D.D.M. Institute of Community Health, Odense University, Institute of Psychology, Aarhus University, and the Municipal School Dentistry Service, Randers, Denmark

ABSTRACT Absorbed lead continuously adds to the lead deposited in the vascular ized circumpulpal dentin of the teeth. Thus, this dental tissue is expected to contain a lead concentration which reflects the integrated lead exposure during the time from completion of tooth formation to tooth extraction or shedding. A method has been developed to assess the lead level in the dentin surrounding the pulp chamber in deciduous teeth. Variation within the tooth is minimal, but upper medial incisors show a slightly lower lead level than do other incisors, as assessed in 714 teeth from first-grade Danish school children. This tendency was not confirmed, however, in a small number of paired teeth from the same children. The new method appears advantageous for epidemiological studies of lead neurotoxicity in children. Introduction used as an integrated measure of the total exposure during early life. Lead levels Determination of lead concentrations may vary, however, between different in shed deciduous teeth has become a tooth tissues and between different tooth useful measure of past lead exposures in types. Thus, the highest lead concentra children. This metal accumulates in cal tions are found in the inner and outer cified tissues owing to the formation of surfaces of the tooth, i.e., the outer layer sparingly soluble lead phosphate. The of the enamel and the circumpulpal development of deciduous teeth starts al dentin.1,6 The circumpulpal dentin, ready in prenatal life, and the average which is formed after the tooth formation lead level in the shed teeth has been has been completed, continues to accu mulate lead until the tooth is shed.6,11 * Presented in part at the 2nd International Con Primary dentin contains much lower lead ference on the Clinical Chemistry and Chemical Tox concentrations. In addition to variations icology of Metals, Montreal, Canada, July 19-22, within individual teeth, the lead concen 1983. tration in whole teeth (or crowns) tends t To whom reprint requests should be addressed at Institute of Community Health, J. B. Winsl0ws to decrease from the medial incisors to Vej 19, 5000 Odense, Denmark. the premolars.2,5 A more extensive report 270 0091-7370/84/0700-0270 $00.90 © Institute for Clinical Science, Inc. LEAD IN CIRCUMPULPAL DENTIN 271 recently concluded that the largest dif ference between incisor lead levels was found when comparing upper medial in cisors to lower lateral incisors.9 How ever, such variations have not been found in less extensive studies of circumpulpal dentin levels.4 The possibility ex ists that the variation related to tooth type could be partly due to a differential proportion of dental tissues in the dif F i g u r e 1. Special vise designed to split deciduous ferent tooth types. teeth into two halves. Three different methods have been used previously: (1) whole tooth or groove is cut vertically in the midline of crown;2,3’5,911 (2) a 300 |xm fragment of the anterior and posterior surfaces by dentin from a 600 |xm thick section of the means of a thin diamond grinder. The tooth;7,8 and (3) circumpulpal dentin ob tooth is then placed in a special vise tained from the root canal with a which splits the tooth into two halves reamer.4’8 Each of these methods may (figure 1). Soft tissue residues are then suffer from drawbacks with regard to removed from the pulp chamber by speed of analysis, inter-tooth variability means of a dental explorer, followed by or biological interpretation. Circum a one-minute immersion in about one ml pulpal dentin attracts particular interest, of 10 percent hydrogen peroxide. Re because only this tissue has a blood maining surface contamination is re supply and, in principle, accumulates moved by subsequent washing of the lead shortly after the time of tooth erup tooth fragment for 20 seconds in an ul tion and until the tooth is shed (or ex trasonic bath with concentrated ethanol tracted). Owing to root resorption prior followed by drying. Clean laboratory to the shedding of deciduous teeth, little techniques are used throughout. The circumpulpal dentin remains apart from dentin bordering the pulp chamber is re the tissue lining the pulp chamber. A moved by a small wolfram carbide ro method has therefore been developed to sette burr.* About one percent of the prepare secondary dentin from the pulp tooth weight, i.e., an average of about chamber of shed deciduous teeth. 0.65 mg, is sampled in this way. The powdered dentin is weighed directly into Materials and Methods a sampling cup, and 100 |jl1 of concen Deciduous teeth are currently being trated nitric acid are added for overnight collected from children in first-grade dissolution. The following day, 400 jjlI of classes of schools in four municipalities redistilled water are added, and the so in Denmark. The 714 teeth used in the lution is analyzed in two independent present study all originated from six to runs by electrothermal atomic absorption seven-year-old children from the city of spectrometry, t Small amounts of dentin Aarhus. This epidemiological study is cause no discernible matrix effect; nitric coordinated by ONH; all teeth were acid is added to all standard solutions. examined and classified by KL, and anal The temperature program for a 20 jxl yses were performed at Odense Univer sample is: drying at 100°C for 25 seconds, sity, Denmark. Only teeth without fill ings were used. Some teeth could be * Borer rund 008, Hager & Meisinger GMBH, D- 4000 Düsseldorf 1, Federal Republic of Germany. split into two parts of similar size by use t Perkin-Elmer model 305A with an HGA-76 of a pointed or sharp instrument (pref graphite atomizer, autosampler AS-1, deuterium erably a mandrel). Otherwise, a small background corrector and recorder. 272 GRANDJEAN, HANSEN, AND LYNGBYE ashing at an increasing (rate 2) tempera TABLE I ture up to 700°C, atomization for three Day-to Day Variation of Micro seconds with gas stop at 2100°C followed Lead Analysis (yg/g) of "Reference Materials" by three seconds at maximum tempera Number of ture. By the technique described, an av Material Analyses Mean + S.D. erage of about 20 teeth can be analyzed per day. IAEA bone* 41 3.09 + 0.41 Tooth powderf 5 9.08 + 2.87 Three different solid “reference mate Hydroxyapatite 47 0.41 + 0.11 rials” were used. A sample of powdered tooth was kindly provided;^ this sample ♦Certified lead level, 3.1 ± 0.28 yg/g; variation of "accepted" levels in interlaboratory compar consisted mainly of particles of 0.1 to 0.5 ison, 3.0 ± 1.08 yg/g mm diameter.10The IAEA standard bone fMean of 13 laboratories, 8.1 yg/g; range, 4.3 - material, § which has a certified lead con 15.5 yg/g (reference 10) centration for aliquots of at least 100 mg, was also used. Inhomogeneity of these materials could cause some analytical in table II indicate that lead concentra variability when using aliquots of about tions decrease with subsequent sam 1 mg. Finally, commercial hydroxyapa- plings into the deeper dentin tissue. titell was used as a low-lead-level, dentin Thus, a homogeneous sample of circum like reference material. pulpal dentin must be as small as allowed by the sensitivity of the detection method. A sample of 0.5 mg seems suf Results ficient, because it will result in 0.02 ng Although day-to-day variations in lead of lead in the 20 |xl of solution injected levels of standard solutions were min into the graphite tube, if the dentin lead imal, larger variations occurred in the level is 1 |xg per g. solid materials (table I). For the bone and The total of 714 shed deciduous teeth tooth materials, some of the variation represents roughly one-half of the teeth may be due to inhomogeneity of the sam collected in Aarhus. A comparison of lead ples. levels in different incisors is shown in The reproducibility of the method was table III. Although the lead levels in assessed by determining the lead level of each of the tooth types varied widely (av circumpulpal dentin prepared indepen erage coefficient of variation, 56 per dently from both halves of each of twelve cent), each group of incisors showed deciduous teeth. The average lead con almost the same average lead level. How centrations varied between 0.54 and 16.6 ever, upper medial incisors showed lead |xg per g, and individual values for half levels which were lower than those of teeth differed by an average of 1.2 n-g per g from the tooth mean, i.e., an average of 10.7 percent, only one value being TABLE I I above 20 percent. Additional studies Lead Levels (yg/g) in Subsequent Layers of concerned five teeth where three sub Circumpulpal Dentin of Five Deciduous Teeth sequent samplings of about one mg from each tooth were made in order to pene First Second Third

trate into deeper dentin layers. The data 3.3 1.6 1.6 4.3 2.2 3.1 5.2 3.1 2.9 $ Dr. M. Stack, MRC Dental Unit, Bristol, U.K. 6.3 1.8 2.1 § Reference Material H-5, IAEA, P.O. Box 100, 9.6 7.2 2.9 1400 Vienna, Austria. II Bio-Rad Laboratories, Richmond, CA 94804. x = 5.7 x = 3.2 x = 2.5 LEAD IN CIRCUMPULPAL DENTIN 273 TABLE I I I indicator of integrated lead exposure Lead Levels (yg/g) in Secondary Dentin Adjacent to during the early years of life when the the Pulpal Chamber of Shed Deciduous Incisors Sampled in 1982 from First-Grade School Children nervous system is particularly suscep in Aarhus, Denmark tible to the toxic effects of lead. Unfor tunately, no uniform approach to tooth Tooth Type Number Mean analysis has appeared. Several authors have recommended the analysis of whole Medial, upper right 100 8.6 left 97 9.5 tooth for lead:2,3,5,9 the procedure is lower right 55 12.5 left 59 10.6 simple, contamination during analysis is Lateral, upper right. 113 11.2 unlikely, and sufficient material is avail left 102 12.5 lower right 89 11. 7 able to obtain results of good precision. left 99 11.8 However, although most shed deciduous teeth only represent the crown, some may occasionally contain root parts, upper lateral incisors (t, = 4.44; p < especially if the tooth has been extracted 0.001) and those of lower medial incisors or if it has been shed at an angle. Also, (t = 3.24; p < 0.005). Within the same interlaboratory variation is quite large.10 tooth types, right and left side teeth Moreover, most of the deciduous tooth showed no statisically significant differ tissue is formed during a relatively short ences. period in early life, and the average lead The differences related to tooth type level in the tooth may not in any accurate were further studied in several pairs of way reflect the lead exposure of the body teeth which originated from the same during the life-time of the child. Based children. Unfortunately, most of these on these considerations, circumpulpal pairs are right-left pairs of the same tooth dentin would appear to be a superior type, because these teeth tend to be tissue to analyze, if the problems of small shed at about the same time. The results aliquots and risk of contamination can be from 26 such pairs are available for ex overcome. amination at this time. One pair of lateral Our method is quite similar to the incisors showed 29.1 |o.g per g in one “reaming” method described by Shapiro tooth and 9.2 |xg per g in the other. Oth et al ten years ago.6 Although the depth erwise, the mean difference from the of penetration with the rosette burr is pair average was 14.1 percent of the pair difficult to standardize, the results in average for 11 medial incisors and 11.7 table II suggest that little variation will percent for 14 lateral incisors (14.5 per be seen when aliquot sizes are kept cent when the aberrant pair is included). below one mg. The reproducibility of the These variations are only slightly larger method is further indicated by the small than the variations found within indi differences found when comparing lead vidual teeth. In addition to the right-left levels in two halves of the same tooth or pairs, six pairs of upper medial-lateral in in two teeth from the same child. cisors were available. In two of these Quality control procedures are partic pairs, the medial incisor was higher in ularly difficult when working with solid lead than was the lateral incisor from the materials with nonhomogeneous distri same child, and the numerical difference bution of the element assessed. The re from the pair mean averaged 14.3 per sults obtained when analyzing the IAEA* cent of the pair means. powdered bone material show the ex pected average, but the coefficient of Discussion variation of 13 percent for our micro Much emphasis has been placed on analysis is higher than the certified nine the use of lead deposition in teeth as an percent for aliquots of at least 100 mg. 274 GRANDJEAN, HANSEN, AND LYNGBYE However, it is much below the coeffi other children and much higher than any cient of variation of 36 percent found in result ever seen in circumpulpal dentin the IAEA interlaboratory comparison in our laboratory. Although the detailed study. Similarly, only a few grains of the exposure history of this child is un relatively coarse tooth powder used in a known, this case may indicate that lead comparison study10 could be analyzed by analysis of circumpulpal dentin as de our new method, and a considerable scribed is indeed a sensitive indicator of variation was found by us, although the past lead exposures. average was as expected. The best ma terial for the purpose of internal quality control is probably commercial hydroxy- Acknowledgments This study has been supported by the Danish apatite, usually employed for column Medical Research Council. The collection of shed de chromatography, especially if an addi ciduous teeth from school children in Aarhus was suc tional product with a higher lead concen cessfully achieved through the efficient cooperation tration can be obtained. with additional members of the research team: Inge Beese, Janne Freitag, Troels Lyngbye, Anegen Tril- The question regarding lead levels re lingsgaard, and Lillian Vangberg. The lead analyses lated to tooth type may not be com in Odense were performed by Ranja Bjerring. pletely solved when using circumpulpal dentin for analysis. Thus, the data in table III suggest that upper medial inci sors contain less lead than do other in cisors. However, this tendency remains References to be assessed in paired teeth from the 1. B r u d e v o l d , F., A a s e n d e n , R., S r in iv a s ia n , B . N., and B a k h o s , Y.: Lead in enamel and sa same individuals. A previous study of liva, dental caries and the use of enamel biopsies one whole set of teeth from an adult in for measuring past exposure to lead. J. Dent. dividual failed to indicate any tooth-type Res. 56:1165-1171, 1977. relationship in circumpulpal dentin.4 On 2. D e l v e s , H. T., C l a y t o n , B. E., C a r m ic h a e l , A., B u b e a r , M., a n d S m it h , M.: An appraisal of the contrary, recent studies of whole the analytical significance of tooth-lead m easure teeth (crowns) have suggested that upper ments as possible indices of environmental ex posure of children to lead. Ann. Clin. Biochem. medial incisors contain more lead than 19-,329-337, 1982. do other incisors.2,5 9Thus, additional in 3. E w e r s , U., B r o c k h a u s , A., W in n e k e , G., formation on the possible variations in F r e ie r , I., J e r m a n n , E ., and K r a m e r , U.: Lead in deciduous teeth of children living in a non- circumpulpal dentin would be needed to ferrous smelter area and a rural area of the FRG. allow a more definite judgment. Int. Arch. Occup. Environ. Health 50:139—151, The diagnostic validity of results ob 1982. 4. G r a n d je a n , R, N i e l s e n , O. V., and S h a p ir o , tained by the present method is difficult I. M.: Lead retention in ancient Nubian and to assess. However, increased lead levels contemporary populations. J. Environ. Pathol. in circumpulpal dentin are no doubt re Toxicol. 2:781-787, 1979. lated to increased lead concentrations in 5. P in c h in , M. J., N e w h a m , J., and T h o m p s o n , R. P. J.: Lead, copper, and cadmium in teeth of the circulating blood. We have had the normal and mentally retarded children. Clin. occasion to analyze an incisor from a Chim. Acta 85:89—94, 1978. 6. S h a pir o , I. M., N e e d l e m a n , H. L., andTuNCAY, seven-year-old boy who has been ex O. C.: The lead content of human deciduous and posed to high lead levels. The blood lead permanent teeth. Environ. Res. 5:467-470, concentration has been about 50 |jug per 1972. 100 ml on four determinations during the 7. S h a p ir o , I. M., D o b k in , B., T u n c a y , O. C., and N e e d l e m a n , H. L.: Lead levels in dentin and previous two years but had decreased to circumpulpal dentin of deciduous teeth of 31 |xg per 100 ml at the time when the normal and lead poisoned children. Clin. Chim. incisor was shed. The circumpulpal Acta 46:119-123, 1973. 8. S h a p ir o , I. M ., M it c h e l l , G., D a v id s o n , I., and dentin showed a lead level of 314 jULg per K a t z , S. H.: The lead content of teeth. Arch. g, about 30-fold above the average of Environ. Health 20:483-486, 1975. LEAD IN CIRCUMPULPAL DENTIN 275

9 . S m it h , M., D e l v e s , T., L a n s d o w n , R., C l a y t o n , rative Interlaboratory Studies in Chemical Anal B., a n d G r a h a m , P.: The effects of lead exposure yses. Egan, H. and West, T. S., eds. Oxford, on urban children: The Institute of Child Health/ Pergamon, 1982, pp. 115-118. Southampton study. Develop. Med. C h ild . 11. St e e n h o u t , A. and P o u r t o is , M.: Lead accu Neurol. 25:1-54, 1983. mulation in teeth as a function of age with dif 10. S t a c k , M. V. and D e l v e s , H. T.: Tooth lead anal ferent exposures. Brit. J. Industr. Med. 38:297- ysis, an interlaboratory survey. IUPAC Collabo 303, 1981.