Elevated T cell subpopulations in dental students

Elevated

T cell subpopulations

D. J. Eedy, M.B., M.R.C.P.,* D. Burrows, T. Clifford, F.D.S.,*** and A. Fay, B.Sc., Royal Victoria Hospital, Belfast, Ireland

in dental

students

M.D., F.R.C.P.,** M.D., M.R.C.Path.****

The absolute numbers of circulating white cells and lymphocyte subpopulations were studied in 26 final-year dental students and compared with a control group of 28 medical students. The total lymphocyte count, total T cell numbers (CD3), T helper/inducer (CD4), and T suppressor/cytotoxic (CD8) numbers were significantly elevated in the dental students as compared with the control group. There was no significant difference in the T helper/inducer to T suppressor/cytotoxic cell ratios or the circulating B cell (CD21) and natural killer cell (CD16) numbers between the study and control groups. Patch testing to mercury and mercuric compounds in both the study and control groups showed no evidence of cutaneous hypersensitivity to mercury. The reason for the observed elevations in T cell subpopulations in dental students is not clear. However, one possible explanation is the dental student’s occupational exposure to mercury. Further work is underway to examine this possible relationship and it is suggested that dental personnel take adequate measures to reduce their exposure to mercury until the results of these studies are available. (J PROSTHET DENT 1990;63:593-6.)

R

eports concerning the health and safety of dentists have appearedsince the 1920sand suggestthat dentists and dental health professionalsare at risk from a wide range of hazards, including exposure to anesthetic gases, mineral dusts, acrylic polymers, mercury, and beryllium.’ Of thesehazards,mercury toxicity in organic and inorganic forms is probably best known and hasbeen the topic of debate and researchin dentistry since the 1830~.~ Although dentists are generally awareof the toxic nature of mercury and avoid handling mercuric compoundsin the preparation of amalgam,they are undoubtedly exposedto higher levels of mercury than the rest of the population, especially when removing old dental amalgam restorations.3p 4Elevated mercury levelshave beenfound in various tissuesin dentists, and a recent study of dentists in Pennsylvania showeda positive correlation between mercury levels in tissue and alterations in nerve conduction, performance in neuropsychological tests, and perceived health symptoms.5*7The development of skin hypersensitivity to mercuric compoundshas been reported, with a progressiveincreasein the development of hypersensitivity to mercury asstudents progressedthrough their dental training.s In 1984, Egglestongdescribed three patients in whom total lymphocyte counts roseafter the removal of amalgam

*Research Fellow, Department of Dermatology. **Consultant Dermatologist, Department of Dermatology. ***Consultant in Restorative Dentistry, Dental School. ****Consultant Immunologist, Immunology Laboratories. 10/1/18380 THE

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restorations and suggestedthat the presence of dental amalgamin restorations may reduce circulating lymphocyte numbers in patients. No subsequentstudies of patients have been doneto confirm this work and there have been no such studies in dental personnel. In an attempt to investigate the possibility of alterations in the immune system brought about by mercuric or other compoundsin the dentist’s environment, we investigated the levels of circulating white cells, lymphocyte subpopulations, and type IV skin hypersensitivity to mercury and mercuric compoundsin dental students.

SUBJECTS AND Study group

METHODS

Twenty-five final-year dental students formed the study group. There were 11 menand 14womenstudents, with an averageageof 21.8 years of age (range 20 to 22 years). The students volunteered after explanation of the study from their final-year tutor, and all of the students who were asked participated in the study.

Controls A control group of 28 medical students was recruited, 18 men and 10 womenwith an averageageof 22 years (range

21 to 23 years). The students were recruited during their dermatology elective, and all of the students who were askedparticipated in the study. Students from both groupswereexcluded if they had any history of skin problems. Students from both groupswere randomly recruited into the study at the same time, and the study was performed over a 3-month period. Blood sam593

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Fig. 1. Lymphocyte counts and lymphocyte subpopulations in study group (dental students) and control group (medical students). Mean x log/1 ? SEM; *, significant difference p < 0.05; **, significant different p = 0.002. pling and patch testing wereperformed on the sameday for each student. All of the students had dental mercury amalgamrestorations.

Determination subpopulations

of lymphocyte

Lymphocyte subpopulations (T, T helper/inducer, T suppressor/cytotoxic, B, and NK cells)weredetermined by use of monoclonal antibodies and flow cytometry. Heparinized blood samples(5 ml), obtained between 9 AM and 10AM and transported immediately to the laboratory, were processedin the following manner: 100~1aliquots of blood were incubated for 15 minutes on ice with 5 ~1 of the respective monoclonal antibodies, OKT3 (CD3) T cells; OKT4 (CD4) T helper/inducer; OKT8 (CD8) T suppressor/cytotoxic, OKB7 (CD21) B cells (Ortho Pharmaceutical Corp., Raritan, N.J.), or 10~1of Leu-7 (CD16) NK cells (Becton Dickinson & Co., Mountain View, Calif.). After two washesin phosphatebuffered saline (PBS) (3 min; 400 gm), 100 J of 1:20 goat antimouse fluorescein isothiocyanate (Ortho Pharmaceutical Corp.) was added to the respective preparations and an additional 15 minutes of incubation on ice was performed. Appropriate controls for background staining were included in eachassay.After two additional washesin PBS, red cells were lysed by use of whole blood quick-stain lysing reagent (Coulter Immunology, Hialeah, Fla.), and the remaining leukocytes were resuspendedin 1% paraformaldehyde-PBS. Analyses were performed on an EPICS 541flow cytometer (Coulter Electronics Ltd). For eachsample,5000lymphocytes were analyzed and the percentageof positive staining cellswasde-

594

termined with the instrument’s immunoprogram.Absolute numbers of the circulating lymphocyte subpopulations were calculated from the total and differential white cell counts.

Patch

testing

Patch testing wasdoneon all subjectsof the study group and the control group for sensitivity to 0.5% mercury in petrolatum, 0.01% phenyl mercuric acetate (aqueous) (Chemotechnic Battery ChemotechniqueDiagnosticsAB, Malmo, Sweden), and 50% amalgam powder in petrolatum.lO*l1 A negative control was included in all cases.The patch test materials were applied to the upper back of the subject under 8 mm rimmed aluminium disks (Finn chambers,Epitest Ltd., Hyryla, Finland) mounted on an acrylate adhesive-coatedpaper (Scanpor tape, Epitest Ltd.) as described by Cronin.12Sites of application of the patch tests were marked. The subjects were askedto remove the patch tests 48 hours after application and the patch tests were read in the clinic within 72 hours.

Statistics Comparisonsbetweenthe groupswere madeby the nonparametric Mann-Whitney U-test and the test statistic (z) and p value given. Values for p greater than 0.05 were judged to be statistically significant.

RESULTS There wasno significant difference in ageor sexbetween subjects in the study group and the control group (z, -0.401; p 0.33).

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T CELL

SUBPOPULATIONS

IN DENTAL

STUDENTS

I. Absolute numbers (XIOg/l) of circulating control group

Table

white cells and lymphocyte subpopulations

Dental students (n = 25) Cell population

White cell count Lymphocyte count* Neutrophila T cell (CDS)? T helper/inducer (CDJ* T suppressor/cytotexic (CDs)*

B cell (CDr1) NK cell (CD& *Significant @ignificant

White

difference difference

Medical students (n = 28)

ii

SEM

x

SEM

5.30

0.24 0.12 1.08 0.09 0.05 0.05 0.01 0.02

5.45 1.47 3.85 1.05 0.56 0.41 0.12 0.15

0.377 0.09 1.58 0.08 0.05 0.04 0.01 0.02

1.87 3.16 1.37 0.72 0.53 0.11 0.15

cell populations

tests

None of the subjectshad positive patch test results recorded in either the study group or the control group.

DISCUSSION In this study a significant increase was shown in the numbersof circulating lymphocytes present in the peripheral blood of dental students compared with medical students. This significant increase was also present in the helper/inducer T cell subsetand the suppressor/cytotoxic T cell subset.The ratio of helper to suppressorcellswasnot significantly different betweenthe two groups.None of the subjects from either group had T cell values outside the referencerange for our laboratory. However, this reference rangewasobtained from a population 18 to 65 years of age and may not be directly comparablewith subjectsin the age range of 20 to 23 years as in the present study. The reasonfor the differencesisfar from clear. The blood sampleswere taken from all of the subjectsbetween 9 AM

TEE

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Normal

range

4.00-11.00 1.40-3.50 2.50-7.50 0.38-1.98 0.18-1.30 0.13-0.93 0.03-0.29 0.03-0.69

at p < 0.05. at p = 0.002.

The results of the white cell counts and lymphocyte subpopulations are shown in Table I and Fig. 1. No significant difference was found between the total white cell count or absolute numbers of neutrophils and monocytes between the study and control groups. However, a significant difference was found between the absolute numbers’of circulating lymphocytes, with a higher number in the study group (z -2.575; p 0.01). A significant elevation was found in the total T cell numbers (CD3) (z -3.068; p 0.002), the T helper/inducer cell numbers (CD4) (z -2.539; p O.Oll), and the T suppressor/cytotoxic cell numbers (CDS) (z -2.041; p 0.0412) in the dental students. No significant difference wasfound in the T helper/inducer to T suppressor/cytotoxic cell ratio (z -0.615; p 0.538) or in the circulating B cell (CD21) (z -0.733;~ 0.464)and NK cell (CD16) (z -0.018; p 0.985) subpopulations betweenthe study and control groups.

Patch

in dental students and

OF PROSTHETIC

DENTISTRY

and 10 AM to eliminate the effect of diurnal variation of T cell numbers.13Although there wasa small preponderance of womenin the study group, the sexdifference betweenthe groupswasnot significant and would be unlikely to account for the differencesobservedin lymphocyte subpopulations between the groups. One report implicated dental amalgamrestorationswith alterations in circulating lymphocyte numbers? However, the alterations were observedin patients, not dentists, and the effect of the amalgamrestorations appeared to be to depresscirculating lymphocyte numbers. Although it has been shown that mercury is released from mercury amalgamrestorationsduring the processes of mastication and grinding, it is not likely that thii is the explanation for the observeddifferences in T cell subpopulations becauseall of the subjects in the study and control groups had amalgamrestorations.‘“rs However, since the number, size, or position of mercury restorations were not correlated with the observed changesin lymphocyte subpopulations in the present study, it is not possible to exclude intraoral mercury as a causative factor. It is tempting to speculatethat the changesin circulating lymphocyte numbers and T cell subpopulations observedin the dental students might be related to exposure to mercury in asmuch as all of the students in this group were regularly placing mercury amalgam restorations. Dentists absorbmercury through contact with the skin and from inhalation, especially when removing deteriorated amalgam restorations.16-18However, further work is required to establish whether mercury can produce the changesin circulating T lymphocytes that were observedin this study. Presently, we are comparing circulating lymphocyte numbers in dental personnelwho are exposedto mercury. The failure to find evidence of skin sensitization to mercury or mercuric compoundsconfirms the view that cutaneous hypersensitivity to mercury in dental personnel, while existing, is probably 10w.s~~~ This low sensitivity 595

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should not be taken to indicate that the observed changes in T lymphocyte subpopulations are not related to mercury exposure, because it has been shown that patients with cutaneous hypersensitivity to nickel do not have alterations in their lymphocyte function20

CLINICAL

SIdNIFICANCE

Although this study is preliminary in nature, it seems sensible for dentists to minimize their risk of exposure to mercury. Ways in which this may be achieved include use of nonamalgam posterior restorations and greater care in removing amalgam restorations, such as using mercury filtration masks and high-suction rubber dam techniques.21-23 We acknowledge the assistance of Mr. A. Crockard, Senior Sciand Miss J. McKirgan SLSO, Immunology Laboratories, for performing the lymphocyte subpopuIation estimations; Dr. J.D. Merrett, Department of Medical Computing and Statistics, The Queen’s University of Belfast, for advice on the statistical analysis; Miss May Weller for preparing the manuscript; and the medical and dental students who volunteered for our study. entist,

REFERENCES 1. Mess& J. Occupational safety and health in the dental work-place. In Goldman HS, Hartman KS, Me&e J. Occupational hazards in dentistry. Chicago: Year Book Medical Publ, 1984;1-19. 2. Berlin MH, Clarkson lW, Friberg LT, et al. Maximum allowable concentrations of mercury compounds. Arch Environ Health 1969;19:891905. 3. Nixon GS, Rowbothan TC. Mercury haxards associated with high speed amalgamators. Br Dent J 1971;131:208-311. 4. Richards JM, Warren PJ. Mercury vapor released during the removal of old amalgam restoratians. Br Dent J 1985$231-2. 5. Wannay A, Sigaerasen J. Mercury accumulation in placenta and foetal membranes: a study of dental workers and their babies. Environ Phys Biochem 1975;5:348-52. 6. Huggins I-IA. Systemic reactions to silver amalgam lillings. Workshop on biocompatihility of metals in dentistry. Chicago: National Institute of Health, American Dental Association, 19&1;201-264.

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of

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7. Ship II, Shapiro IM, Miller WD. Mercury poisoning in dental practice. Compend Contin Educ Dent 1983;4:107-10. 8. White RR, Brandt RL. Development of mercury hypersensitivity among dental students. J Am Dent Assoc 1976;92:1204-7. 9. Eggleston DW. Effect of dental amalgam and nickel alloys on T lymphocytes: preliminary report. J PROSTHET DENT 1964;15:617-23. 10. Axe11 T, Bjorkner B, Fregert S, Niklesson BO. Standard patch test series for screening of contact allergy to dental materials. Contact Dermatitis 1983;9:82-4. 11. Axe11 T. Evaluation of a preliminary standard patch teat series for the screening of dental materials. Contact Dermatitis 1983;9:86-7. 12. Cronin E. Contact dermatitis. Edinburgh: Churchill Livingstone 1980;119. 13. Signore A, Cugini P, Letizia C, Lucia P, Murano G, Poxzilli P. Study of the diurnal variation of human lymphocyte subsets. J Clin Lab Immuno1 1985;17:25-8. 14. Abraham JE, Svare CW, Frank CW. The effect of dental amalgam restorations on blood mercury levels. J Dent Res 1984;63:71-3. 15. Gray DD, Cox RD, Reinhardt JW. Chewing releases mercury from fillings. Lancet 1979;i:985-6. 16. Joselow M, Goldwater L, Alvarel A, Herndon J. Absorption and excretion of mercury in man. XV. Occupational exposure among dentists. Arch Environ Health 1968;17:39-43. 17. Hursh JB, Clarkson TW, Cherian MG, Vostal JJ, Vander Malie R. Clearance of mercury vapor inhaled by human subjects. Arch Environ Health 1976;31:302-9. 18. Svare CW, Frank CW, Chan KC. A quantitative measurement of mercury vapour emission from setting dental amalgam. J Dent Res 1973;52:740-3. 19. Burrows D. Hypersensitivity to mercury, nickel and chromium in relation to dental materials Int Dent J 1986;36:30-4. 20. Al-Tawil NG, Marcusson JA, Moller E. T and B lymphocytes in patients with nickel sensitivity. Stand J Immunol 1985;22:495-502. 21. Status report on posterior composites. Council on dental materials, instruments and equipment. J Am Dent Assoc 1983;107:74-5. 22. Mantyla DG, Wright OD. Mercury toxicity in the dental office: a neglected problem. J Am Dent Assoc 1976;92:1189-94. 23. Recommendations in dental mercury hygiene. Reports of Councils and Bureaus. J Am Dent Assoc 197&96:487-g. Reprint

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DR. D. J. EEDY DEPARTMENT OF DERMATOLOGY ROYAL VICTORU HOSPITAL BELFAST BT12 6BA IRELAND

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