O022-3565/95/2721-0264$03.

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THE Jouai. o Paaucowoy um Expaswmi. Tissiui’n.mcs voL 272, No. 1
Copyright C 1995 by The American Society for Pharmacology and Experimental Therapeutics Printed in USA
,mET 272:264-274, 1995

Sodium 2,3-Dimercaptopropane-1 -Sulfonate Challenge Test for

Mercury in Humans: II. Urinary Mercury, Porphyrins and
Neurobehavioral Changes of Dental Workers in Monterrey,
Mexico1

DIEGO GONZALEZ-RAMIREZ, RICHARD M. MAIORINO, MIGUEL ZUNIGA-CHARLES, ZHAOFA XU,

KATHERINE M. HURLBUT, PABLO JUNCO-MUNOZ, MARY M. APOSHIAN, RICHARD C. DART,
JOSE HORACIO DIAZ GAMA, DIANA ECHEVERRIA, JAMES S. WOODS and H. VASKEN APOSHIAN
Depattment of Pharmacology, Centro de Investigacion Biomedica Del Noreste, Instituto Mexicano Del Seguro Social, Monterrey, Mexico
(D.G.R., Departments
M.ZC.); of Molecular and Cellular Biology (R.M.M., ZX., M.M.A, H.V.A.) and Pharmacology, University of Arizona,
Tucson, Arizona (H.V.A.); Rocky Mountain Poison Center, Denver, Colorado (K.M.H., R.C.D.); Department of Pharmacology, Division Ciencias
de Ia S&ud, ITESM, Suc Correso “J” and (P.J.M.) Cuauhtemoc y Famosa Clinic, Monterrey, Mexico (J.H.D.G.); Battelle SeaWe Research
Center (D.E., J.S.W.) and Department of Envimnmentei Health, University of Washington, Seattle, Washington (D.E., J.S.W.)
Accepted for publication September 6, 1994

ABSTRACT
The sodium salt of acid
2,3-dimercaptopropane-1
-sulfonic DMPS administration. This was not so if the urinary mercury
(DMPS) challenge test (300
mg p.o. after an 1 1 -hr fast) was level before DMPS administration was compared with the un-
given in Monterrey, Mexico to dental and nondental personnel. nary coproporphynin concentration. The urinary mercury level
Unne samples were collected and analyzed for total mercury. after DMPS administration is a better indicator of exposure and
The mean mercury urinary excretion (± S.E.) for 6 hr before and renal mercury burden than is the mercury level measured in the
6 hr after DMPS administration for 10 dental technicians, who unne before DMPS is given. Regression analysis showed that
formulate amalgam, was 4.84 p.g ± 0.742 and 424.0 g ± 84.9; the coefficient of urinary mercury was statistically and ad-
for 5 dentists, who use amalgam in their practice, 3.28 pg ± versely associated with complex attention (switching task), the
1 .1 1 and 1 62.0 p,g ± 51 .2; and for 1 3 nondental personnel, perceptual motor task (symbol-digit substitution), symptoms
0.783 L9 ± 0.189 and 27.3 tg ± 3.19. The urinary copropor- and mood. The easily performed DMPS-mercury challenge test
phyrin levels before DMPS administration, which are indicative is useful for monitoring dental personnel for mercury vapor
of renal mercury content, were quantitatively associated with exposure.
the urinary mercury levels among the three study groups after

DMPS has been distributed and used in Europe since 1976. ala or metalloids (Aposhian, 1983; Kemper et al. , 1990). The
In fact, as stated by Schiele et al. in 1989, DMPS “has been first suggestion for the use of DMPS as a provocative or
the approved treatment of choice for more than ten years in challenge test for mercury was made by Osinska and Pro-
West Germany and is valued for its negligible side effects.” janowska (1981) and in the western world by Cherian et al.
Its therapeutic uses have been reviewed (Aposhian, 1983; (1988). Before this time, DMPS was first made available by
Kemper 1990; Aposhian
et al. , et al. , in press, 1995). Phar- the Soviets to American toxicologists working with Iraqi phy-
macokinetic studies of DMPS, p.o. and i.v., in humans have sicians who were treating people who had eaten bread pro-
been performed (Maiorino et al., 1991; Hurlbut et al., 1994) pared from grain seeds that had been treated with a mercu-
and DMPS has been used to treat humans intoxicated with ry-containing fungicide (Clarkson et al., 1981). Since then, it
mercury (Campbell et al. , 1986), arsenic (Gerhard et al., has been used in humans by physicians in attempts to ap-
1992), lead (Chisoim and Thomas, 1985) or other heavy met- proximate the body burden of mercury before beginning che-
lation “therapy” (Gerhard et al. , 1992; Schiele et al. , 1989).
Received for publication June 9, 1994.
The use ofDMPS to treat bona fide mercury toxicity is well
1 Tk work was supported in part by the Superfund Basic Research Pro-
established and accepted (Campbell et al. , 1986; Schiele et
gram, National Institute of Environmental Health Sciences, grant ES-04940
and in part by grant number ES-04696 from the National Institute of Envi- al. , 1989). The use of a provocative or challenge test to esti-
ronmental Health Sciences, National Institutes of Health. mate the body content or exposure to a heavy metal is well

ABBREVIATiONS: DMPS, sodium 2,3-dimercaptopropane-1 -sulfonate; SAT, simple reaction time; IMSS, Insituto Mexicano del Seguro Social;

HPLC, high-performance liquid chromatography.

264
1995 DMPS-Mercury Challenge Test 265

established in medicine. For example, calcium disodium definition of acute versus chronic effects, criteria of exposure
EDTA has been used as a provocative test for lead for many or reliable dose-effect information. Thus, a threshold effect
years but recently has been CriticiZed (Chisoim, 1987). The level has yet to be established, although efforts on the part of
rationale and basis for the test is essentially that, over a several researchers have led to proposed biological threshold
certain range, the urine and blood concentration oflead with- limit values of 25 or 50 p,g/liter of urine (Miller et al. , 1975;
out calcium disodium EDTA treatment may not be suffi- World Health Organization Study Group 1991; Roels et al.,
ciently indicative ofthe body’s lead burden. Although calcium 1985).
disodium EDTA mobilizes lead, it is not an effective mobilizer Dental technicians are an excellent population for the
of mercury and is thus useless as a mercury challenge test. study of threshold effects because they are more homoge-
Aposhian et al. (1992) concluded that after p.o. adminis- neous than other exposed populations with respect to manual
tration of 300 mg of DMPS as a challenge test, urinary dexterity, education, training and test-taking ability. Their
mercury excretion was increased substantially and that col- use as the study population of choice in the present study
lege students with dental amalgams in their mouth excreted amplifies the possibilities of detecting subtle subclinical ef-
approximately three times more mercury in their urine than fects of neurobehavior. The present study was expanded to
those without amalgams. evaluate neurobehavioral performance in 10 mercury-ex-
Urinary porphyrin measurements have been proposed as a posed dental technicians and 13 unexposed medical techni-
biomarker of mercury exposure (Woods et al. , 1991). Porphy- cians.
rims are formed as intermediates in the biosynthesis of heme The purpose of the present research was to determine
in essentially all tissues. In humans and other mammals, five whether urinary mercury levels of dental workers in a devel-
structurally distinct porphyrins, characterized by side chains oping country were within the normal range. Urine samples
with four to eight carboxyl groups, are excreted in the urine of dental workers employed in a modern, new hospital/clinic
in a well established pattern. Prolonged exposure of animals in Monterrey, Mexico and those of a nondental control group
to mercury as methyl mercury hydroxide produced a change were collected under standardized conditions before and up
in the urinary porphyrin excretion pattern. There was as to 6 hr after DMPS administration and analyzed for total
much as a 30-fold increase ofthose porphyrins with four and mercury and porphyrins. Finally, to examine the relation-
five carboxyl groups (Woods et al. , 1991; Bowers et al. , 1992) ships between urinary mercury levels and health status,
plus the excretion ofan atypical porphyrin (“precoproporphy- neural behavioral performance was evaluated.
rin”). The latter is eluted on HPLC between the 4- and
5-carboxyl porphyrins. Similar findings have been reported
for human subjects occupationally exposed to mercury vapor
Methods
(Woods et al., 1991; Bowers et al., 1992). The change in the Clinical. Each subject underwent a history and physical exami-
porphyrin excretion pattern is attributed to Hg , which is nation; female subjects had a urine pregnancy test performed; and
produced by the oxidation of Hg#{176} in the body. Hg inhibits informed consent was obtained before enrollment in the study. Par-
porphyrin metabolism at the site of coproporphyrinogen uti- ticipants were required to refrain from eating seafood for 15 days
lization and promotes oxidation of reduced porphyrins (por- before DMPS administration. The physical examination was re-
phyrinogens) in kidney cortical cells (Woods et al., 1991; peated, vital signs were monitored and blood was obtained for chem-
iced analysis (SMAC 20 and complete blood count) immediately be-
Bowers et al., 1992). Changes in porphyrin excretion proffles
fore and 6 hr after DMPS administration. CliniCal laboratory studies
are highly correlated with both the dose and time course of were performed at the Cuauhtemoc y Famosa Clinic, Monterrey,
mercury exposure (Woods et al. , 1991). Therefore, urinary N.L. Mexico. Subjects were encouraged to void immediately before
porphyrin levels, specifically those of 4- and 5-carboxyl por- and up to 6 hr after DMPS administration and to drink sufficient
phyrins and of precoproporphyrin, may be viewed as a water to maintain a urine output of approximately 500 ml per col-
bioindicator both of mercury exposure and of renal mercury lection period. Informed written consent was obtained. The experi-
content. mental protocol was approved by the Human Subjects and Ethics
The occupational effects of elemental mercury (Hg#{176}) expo- Committees ofthe IMSS and the Human Subjects Committee of the
sure on human behavior and mood have long been recognized University of Arizona. Because DMPS is an investigational drug in
the United States, the study was performed under the Food and
(Neal et al., 1941). Recent information concerning dental
Drug Administration investigational new drug application number
amalgams has made mercury exposure thresholds for ad-
34,682.
verse behavior, mood and symptoms of increasing impor-
PrOtOCOL Before DMPS administration, each participant filled
tance in regard to risk assessment and public health. In a out a consent form, a computerized symptom checklist, a Profile on
1991 national sample of dentists in the United States, the Mood Scale, a brief medical and occupational questionnaire and the
average urinary mercury excretion was 5 pg/liter (Naleway et behavioral test battery. Test administrators were blind with respect
al., 1991). This does not necessarily mean that the mercury to exposure status. Subjects were not informed oftheir mercury level
burden of such professionals is within the “normal” range. and test scores until the end of their participation.
Small but statistically significant changes as to behavior, For the DMPS mercury challenge test, the subjects were fasted
mood and symptoms among dentists at mean urinary mer- overnight beginning 11 hr before DMPS administration to 4 hr after
cury concentrations of36 pg/liter have been reported (Echev- DMPS administration, at which time a turkey sandwich was eaten
(table 1).Urinewascollectedfrom-llto0hrandfrom0to6hr
erria et al. , in press, 1995). Four previous dental studies have
after administration ofthe chelating agent. (The mercury content of
reported health effects associated with urinary mercury 1ev-
the 11-hr urine was determined and then divided by 11 and multi-
els below 50 p.g/liter (Shapiro et al. , 1982; Uzzell and Oler, plied by 6 to obtain the -6 hr or pre-DMPS urinary mercury value).
1986; Ngim et al. , 1992; Echeverria et al. , in press, 1995). Molin et al. (1991) reported a close correlation (r = 0.99) between
Unfortunately, these four studies used diverse epidemiologic urinary mercury excretion during the first 6 hr after DMPS, and that
methods with major weaknesses in recruitment of subjects, during the 24-hr period after DMPS. DMPS, 300 mg p.o. was given
266 Gonzalez-Ramirez et al Vol. 272

TABLE 1 Symptom, medical and work history questionnaires. The

DMPS challenge test protocol symptom questionnaire included measures of physiologic, psycho-
logic and somatic symptoms that have been reported after acute and
-1 1 hr Begin fasting chronic exposures. It was based in part on the Swedish “Question-
Begin overnight urine collection naire 16” (Hogstedt et al., 1985) and questionnaires used in previous
0 hr End overnight urine collection
central nervous system studies of mercury (Albers et al., 1988; Kal-
Collect blood sample
Administer 300 mg of DMPS p.o.
lenbach 1988), organic solvents (Echeverria et al., in press, 1995) and
Begin 0-6-hr urine collection lead (Baker et al., 1984).
+4 hr End fast One component of the questionnaire evaluated conditions that
+6 hr End 0-6-hr urine collection might influence test performance. The variables ofinterest included
Collect blood sample age; race; gender; education; eyeglass wear; income; and medical
history of neurologic disorders, hypertension, diabetes and use of
medications. Questions regarding personal habits included a de-
and subjects were encouraged to drink sufficient mercury-free water tailed history ofthe use ofalcohol, caffeine and nicotine and chemical
to maintain a urine output of approximately 500 ml per collection exposures received from hobbies.
period. The DMPS dose was chosen on the basis of previous studies This information supplemented an occupational history and work
at the University of Arizona (Aposhian et al., 1992; Maiorino et al., practice questionnaire that covered sources of mercury exposure
1991) and other clinical reports (Kemper et al., 1990). This dose was such as the number of operatories in one clinic; the number of
given to each subject independent of the body weight because the
amalgam placements, removals and spills per week; the number of
regimen is a diagnostic test.
amalgams each subject had in his or her mouth; and consumption of
Urinewas collected in acid-washed 3-liter polyethylene collectors
seafood.
(Baxter, McGaw Park, IL) and the volume was measured in plastic
Mood and vocabulary. An evaluation of mood (Proffle of Mood
graduated cylinders. Concentrated HC1 was immediately added to
States; McNair et al., 1971) was included because such effects have
give a final concentration of 1.8%.
The acidified urine was trans-
been consistently reported in workers with mercury concentrations
ferred to polyethylene bottles, frozen by placement in a dry-ice con-
between 30 and 100 gIliter ofurine (Roels et at., 1985; Fawer et al.,
tamer and stored frozen until analyzed. All glassware or plastic ware
1983; Zedda et al., 1980; Piikivi et al., 1984; Soleo et al, 1990).
was soaked in 2% nitric acid at least overnight or was washed with
Behavioral test battery. These tests of digit-span (Smith et al.,
20% nitric acid. All urine samples were digested, at least, in dupli-
1983), symbol-digit substitution (Baker et al., 1985), simple reaction
cate and three cold vapor atomic absorption determinations were
made on each digest. The amalgam score was determined for each time (Baker et al., 1985), the ability to switch between tasks (Tou-
tonghi et al., 1991) and verbal ability (Baker et al., 1985) were based
subject. It is a measure of the diameters of all the amalgam surfaces
on all the teeth in a subject’s mouth. It has been defined more on previous results found among mercury-, lead- and solvent-ex-
specifically and a more detailed determination of it has been de- posed cohorts. To assess potential deficits in manual dexterity, the
scribed previously (Aposhian et al. , 1992). one-hole test (Salvendy 1975), a validated manual dexterity task, an
Study design for neurobehavioral testing. Before the DMPS improvement on the traditional Purdue peg board test, was selected.
challenge test, a cross-sectional behavioral evaluation was conducted This test independently assesses component tasks such as the time it
to assist in the interpretation of the health significance of urinary takes to grasp, move, position and reach while transferring small
mercury levels. The primary hypothesis was that mercury-exposed pins from a large target to a small target hole. The test has been
subjects would demonstrate mild subclinical effects associated with found to be sensitive to other neurotoxicants such as toluene and
recent and cumulative body burden of mercury with the urinary ethanol (Echeverria et al., 1991) and in previously exposed mercury
mercury values after DMPS administration as the criteria of burden. workers (Echeverria et al. , in press, 1995). Tremor was not included
It is difficult to measure adverse subclinical nervous system effects because of the short test development time.
because of large individual variation in most functions and the Data analysis. Multivariate regression (SAS-PC, SAS Institute,
number of individual factors that may affect these functions. The Cary, NC) techniques were used to test the hypotheses that recent
process of standardization across behavioral studies is still a re- and chronic measures of mercury exposure were associated with
search question but considerable has beenprogress
made in the adverse changes in symptoms, mood and behavioral function. All
design of such studies, in the use of control
measures and in the test continuous variables used in the analysis were graphically reviewed
measures. The present study was designed to be consistent with (AXUM technical graphics and data analysis, Timetrik, Seattle, WA)
several guiding principles that would maximize the detection of to evaluate their approximation to a normal distribution. Variables
small changes in performance among a small population. First, the with poor approximations of a normal distribution or with extreme
study population itself provided a high degree of uniformity, which values that might dominate the analysis were log transformed. Re-
reduced background noise and amplified the possibility of detecting cent exposure was evaluated in a continuous dose-effect manner with
subclinical effects. Second, comparisons were made with a carefully the use of the concentration of mercury in the urine. The chronic
chosen control group that had comparable, but not identical, techni- index was also evaluated as a continuous variable. A P value of < .1
cal training. Third, the study relied on previously validated, reliable was considered marginally associated and a P value of < .05 was
and well characterized measures of the central nervous system, considered statistically significant. Age in years, race, gender, in-
which were based on previous clinical epidemiologic study results. To come, use of eyeglasses, alcohol consumption (a five-level scale based
improve standardization and quality control and reduce potential on frequency of drinking per week), vocabulary scores, medications,
interviewer bias, we chose to use previously validated computerized a medical history of neurologic importance and use of nitrous oxide
tests drawn from the World Health Organization recommended list were each evaluated as potential confounders. Only the most impor-
of tests, which have been translated into Spanish. tant confounders were retained in the final analysis because of the
Subjects. Thirteen unexposed laboratory technicians and 10 mer- small number of subjects.
cury-exposed dental technicians were recruited from the IMSS lab- For analysis of mercury excretion and porphyrin excretion, single
oratory and the participating dental clinic. The five dentists were factor analysis of variance or Student’s t test was used to determine
omitted from the neurobehavioral analysis because the number who significant differences. A confidence interval of 95% was considered
volunteered was small and the dentists were not comparable in to be statistically significant (P .05). Linear regression analyses
socioeconomic status, education and training with the dental tech- were performed with a general purpose statistics package acquired
mcians or the nondental controls. from Microsoft, Inc.
1995 DMPS-M.rcury Challenge Test 267

Analytical procedures. The urinary mercury levels were deter- The control, nondental subjects were employed at the Cen-
mined by cold vapor generation-atomic absorption procedures, as tro de Investigacion Biomedica Del Noreste, IMSS, in
previously described (Aposhian et al., 1992). The procedure was Monterrey. Dentistry is not practiced at this IMSS facility
again validated immediately before doing the present assays by and research using mercury is nil. The alcohol consumption
analyzing freshly voided urine from a normal individual to which
of this population was small. More than 65% of the subjects
mercury nitrate had added to final concentrations
been ofO.50, 5.0 or
drank alcohol less than once per month (table 2).
30.0 ng/ml, in duplicate, according to the procedure described by
Clinical. Three subjects had mild headaches, one felt
Aposhian et ci. (1992). After a correction for the mercury content of
the urine before the spike was added, six determinations ofthe 30.0 weak and one felt somewhat lightheaded, all of which effects
ag/nil of Hg gave a mean of 29.9 ng/ml of Hg (range, 29.8-30.0), six were thought to be related to the prolonged fast. Two subjects
determinations ofthe 5.00 ng/ml of Hg gave a mean of 4.52 ag/mi of felt nauseous and two others reported symptoms of reflux
Hg (range, 4.51-4.53) and six determinations of the 0.500 ng/ml of and belching. These symptoms may have been attributable to
Hg gave a mean of 0.418 ag/mi of Hg (range, 0.382-0.454). The DMPS or to the carbonated water the subjects consumed.
mercury content of urine samples was analyzed both at the Univer- One subject reported nausea and two episodes of diarrhea;
sity of Arizona in Tucson and the IMSS Biomedical Research labo- his symptoms were thought to be drug related. No other
ratories in Monterrey, Mexico.
adverse signs developed. Slight differences in several blood
Urinary porphyrin analysis, Urine was collected and acidified
levels were observed before and after DMPS administration,
as described previously and then a 35-nil aliquot was dispensed into
without accompanying clinical effects (table 3).
50-tul polypropylene bottles, frozen and sent to Seattle by overnight
express delivery service. Porphyrin analysis was performed on a Urinary excretion of mercury. The administration of
10-mi aliquot, as previously described (Woods et al., 1991; Bowers et the DMPS challenge to dental technicians, dentists and non-
al., 1992). Briefly, the acidified urine was applied to a C-18 solid- dental personnel resulted in an 88-fold (range, 11-335), 49-
phase extraction column (Waters Associates, Milford, MA) and fold (range, 45-76) and 35-fold (range, 14-132) increase, re-
washed with a 35% methanol and 65% sodium phosphate buffer, pH spectively, in the mean 6-hr urinary excretion of mercury
3.5, to remove interfering substances (Bowers et a!., 1992). Porphy- compared with that during the 6-hr period before the admin-
Tins were eluted with 100% methanol; the methanol was then evap- istration of this chelating agent (table 4). These increases
orated and the samples were reconstituted in 0.5 ml of 1 N HC1. were statistically highly significant (table 4). The mean urn-
Porphyrins were separated and quantified by HPLC with a Waters
nary mercury excretion before DMPS administration for the
HPLC system that was equipped with an Econosphere (Alltech/
dental technician group and dentists was 6 times and 4 times
Applied Science, Deerfield, IL) C-18 column. Sodium phosphate (50
mM, pH 3.5) was used as the starting mobile phase and individual greater, respectively, than that of the nondental controls.
porphyrin congeners were eluted with a linear gradient of increasing After DMPS administration, however, the mean urinary
methanol concentration. Porphyrins were measured by their fluores- mercury for the dental technician group and dentists was 15
cence intensity (excitation wavelength, 395 am; emission wave- times and 6 times greater, respectively, than that of the
length, 620 am) with a Shimadzu (Columbia, MD) RF-535 detector. nondental controls.
Porphyrin identification and concentrations were determined by Because toxicologists are usually more concerned
with con-
comparison with a standard curve of authentic porphyrin isomers centrations, the level in micrograms per liter of
of mercury
(Porphyrin Products, Logan, UT).
urine was also calculated (table 4). The urinary mercury
concentration also showed a substantial (at least 12-fold)
increase after DMPS administration. In addition, such data
Results
indicate that the difference in urinary concentrations of mer-
Study population. The subjects were 5 dentists (1 female cury is not as marked as the difference in absolute amounts
and 4 males; age range, 26-34 years;
mean, 30.2 years); 10 that were excreted during the 6-hr period after DMPS.
female dental technicians (age range, 17-27 years; mean, Was there a strong positive correlation between the mean
20.2 years) whose duties included the formulation of amal- urinary mercury level before and after DMPS administra-
gam; and 13 nondental personnel (5 females and 8 males; age
range, 23-51 years; mean, 36.4 years). All dental personnel TABLE 2
were employed in a dental clinic in Monterrey, Mexico and A comparison of demographic factors associated with mdlcal
routinely worked with amalgam. The dental clinic is part of technicians occupationally unexposed and dental technIcians
a hospital built by a progressive company to provide im- occupatIonally exposed to mercury
proved medical and dental care for its employees. The build- Values are mean ± S.D.
ing was less than 5 years old at the time this study began and Unexposed Exposed
has excellent modern medical and dental facilities. The den- Numberofsubjects 13 10
tal clinic has four exsimining rooms and one very small win- Age(yr) 35±8 22±8
dowless preparation room. It is in the latter that a dental Male 8 0
technician prepares dental amalgam as needed. To do this,
Income (new pesos)
<10,000 11 7
the technician removes metallic mercury from a bottle and <20,000 2 2
places it on a small piece of white filter paper and then adds <30,000 1
to it a portion of alloy powder. The technician then carries Without glasses 5 7
this to the dentist in the examining room who squeezes the Alcoholic dnnks/wk 3 0
Frequency
excess mercury with his or her hands through the ifiter paper
1-2days 4
and uses the amalgam to complete the restoration. This <1/wk 1
method of amalgam preparation was used in the United <1/mon 8 10
States about 20 years ago. It has been replaced in the United Medication user 3 1
States by capsules that contain amalgam.
Vocabulary scores 18 ± 3 16 ± 3
268 Gonzalez-Ramlrez et al Vol. 272

TABLE 3
Changes in blood chemistries after DMPS adminIstration
The paired t test was used.
Before DMPS (mean ± SE.) After DMPS (Mean ± SE.) Percent Change P Value
Leukocyte count (x106/mm) 7.6 ± 0.4 8.5 ± 0.4 +12 .0005
Potassium (mEq/liter) 4.3
± 0.1 3.9 ± 0.1 -1 1 <.005
Direct bilirubin (mg/dl) 0.03 ± 0.01 0.05 ± 0.01 +67 <.005
Phosphorus (mg/dl) 3.2 ± 0.1 3.5 ± 0.1 +9 <.005
Triglycerides (mg/do 101.0 ± 16.0 112.0 ± 16.0 +11 <.05

TABLE 4
UrInary mercury level before and after DMPS challenge test
P values determined by single-factor analysis of variance. For unary Hg before and after treatment, P < .001 for the dental technicians, P < .015 for the dentists and
P < .001 for the nondental personnel. For -6 to 0 hr, dental technicians vs. nondental, P < .001 ; dentists vs. nondental, P < .005; dental technicians vs. dentists, P
= .252; ForO to +6hr, dental technicians vs. nondental, P < .001; dentists vs. nondental, P < .001; dental technicians vs. dentists, P = .0597.
Mercu ry level ± SE. Mercury Concentratlon ± S.E.
Group n
-6 to 0 hr (before) 0 to +6 hr (after) -6 to 0 hr (before) 0 to +6 hr (after)
Lg g/1iter
Dental technicians 10 4.84 ± 0.742 424.0 ± 84.9 29.7 ± 6.73 481 .0 ± 121.0
Dentists 5 3.28 ± 1 .1 1 162.0 ± 51 .2 19.8 ± 7.19 275.0 ± 107.0
Nondental 13 0.783 ± 0.189 27.3 ± 3.19 3.00 ± 0.620 37.2 ± 15.1

tion? Although there was a strong positive correlation for the dental technicians (r = 0.051) or for the nondental controls (r
urinary mercury levels ofthe dentists before and after DMPS = 0.473). Although there was a high correlation in the case of
administration (r = 0.996, fig. 1), this was not the case for the the dentists and a poor correlation for the dental technicians,

12oo. (B)DENTALTEHS

t300 Iooo U

8oo
Ca 200 (0
g600 UU U
%iso r = 0.0512
a
400- U
100
200 U U U

a 0 -

012345678 . 0 2 4 6 8 10
tg URINARY Hg; -6 TO 0 HR BEFOREDUPS tg URINARY Hg; -6T00 HR BEFORE DMPS

Fig. I . There was a strong positive cor-

relation between urinary mercury level
before and after DMPS administration
for the dentists but not for the dental
technicians and controls.

w
I:
=
Ca

g
a
=

z
: 0.5 1.0 1.5 2.0 2.5 3.0

0 2 4 6 8 10
P1 URINARY Hg; -6 TO 0 HR BEFORE DMPS I.Lg URINARY Hg; -6 TO 0 HR BEFORE DMPS
1995 DMPS-Mercury Challenge Test 269

we cannot explain this. It is possible that the results with the Associations between behavior, mood, symptoms
dentists might be different ifthere had been a larger number and post-DMPS mercury levels. The associations between
of them. urinary mercury levels and health outcomes are described in
Urinary porphyrin excretion. In the present studies, tables 7 and 8. The urinary excretion of mercury both before
urinary porphyrin concentrations were measured for two rea- and after DMPS administration was associated with adverse
sons. First, we wanted to see whether they correlated posi- effects but the calculated index of cumulative mercury expo-
tively with the burden ofmercury, as indicated with or with- sure based on work histories alone was collinear with age and
out the DMPS challenge test, for subjects occupationally was not useful as an indicator of potential adverse effects.
exposed to mercury vapor. Second, we wanted to determine Based on the urinary excretion of mercury after the DMPS
whether the concentrations ofurinary porphyrins were also a challenge, multiple-regression analyses found that the time
measure of the efficacy of DMPS in promoting mercury ex- to match symbols with digits (digit-symbol substitution) and
cretion from the kidney. the time to switch between tasks (the switching task) were
Correlations were examined between urinary coproporphy- increased and thus adversely affected by exposure to mer-
rin and urinary total mercury among the three groups of cury (table 7). The simpler attention task, which calculated a
study subjects before or after DMPS challenge (table 5). subject’s digit span, decreased in the expected direction by
Linear-regression correlation analyses were performed to one-third ofa digit but did not achieve statistical significance
compare different parameters of urinary mercury and uri- (table 7). Thus, between the two attention tests, the more
nary porphyrins such as concentration (in micrograms per complex task was affected by exposure. The two tests with a
liter), amount (micrograms), nanograms per milligram of strong manual motor component, SRT and the one-hole test,
creatinine and pre- (before DMPS) and postadministration were not significantly affected by mercury exposure even
(after DMPS). The best correlations were obtained when the though the SRT response time increased by 46 msec in the
urinary coproporphyrin concentration (in micrograms per II- expected direction.
tar) before DMPS was compared with the urinary total mer- All the mood scales (table 8) deteriorated with exposure
cury amount (in micrograms) after DMPS (dental technician, but only the differences in scales for tension, anger and
r = 0.921; dentists, r = 0.667; nondental personnel, r = confusion were statistical significant. Reported symptoms
-0.642). There was insufficient data on pentacarboxylpor- related to increased headaches, increased emotionality, poor
phyrin and coproporphyrin levels to do linear-regression cor- comprehension and poor coordination were also associated
relation analyses. Changes in the urinary porphyrin levels with increased urinary mercury excretion.
were examined among the three groups before and after the
DMPS challenge (table 6). After DMPS administration (table
6), there was a decline in the urinary concentrations of each
Discussion
porphyrin compared with the pre-DMPS levels except for the There are a number of results of the present investigation
precoproporphyrms ofthe nondental controls. Although most that warrant discussion, namely DMPS-Hg challenge test,
of these declines were not statistically significant (P > .05), urinary porphyrins and neurobehavioral testing.
perhaps because of the wide variability in porphyrin excre- First, the DMPS-Hg challenge test indicated that the body
tion rates and the relatively small number ofsubjects in each stores of mercury are larger in dental personnel in a modern
group, the trend was consistent. Coproporphyrin had the dental clinic in Monterrey, Mexico than in nondental person-
most substantial decline in this respect, decreasing to 66%, nel who have not been occupationally exposed to mercury
68% and 48% of the before-DMPS values among the dental (table 4). At the occupational level, elemental mercury vapor
technicians, dentists and nondental controls, respectively. is the main form of mercury exposure. Skerfving (1991)
This suggests substantial depletion of renal mercury after stated that the largest group of such workers are dental
DMPS treatment. professionals but that, in Sweden, their exposure is low, with
TABLE 5
Unear-rgression correlation (r) analysis of urinary mercury and coproporphyrin

Dental Technicians Dentists Nondental

Coproporphyrin Total Hg
r r r
p9/liter (pre) L9 (post) 0.921 0.667 -0.642
Mg/liter (pre) p.9 (post - pre) 0.921 0.666 -0.642
g/lfter (pre) Lg/liter (post) 0.977 0.0281 -0.716
Mg/liter (pre) L9/lIter (post - pre) 0.981 0.00728 -0.710
,A9 (pre) ,.i.9 (post) 0.464 0.427 -0.333
L9 (pre) g.g/liter (post) 0.391 -0.256 -0.378
ng/mg of creatinine (pre) ng/mg of creatinine (post) -0.350 0.164 -0.573
.g/liter (pre) L9 (pre) 0.152 0.706 -0.250
pg/liter (post) ,.L9 (post) 0.366 0.636 -0.220
Lg/liter (pre) p.giliter (pre) -0.0304 0.296 0.191
IL9/liter (post) p.g/Ilter (post) 0.338 0.912 0.0904
p9 (pre) p9 (pre) 0.376 0.474 0.01 51
L9 (post) IL9 (post) 0.291 0.441 -0.147
L9 (pre) &g/liter (pre) -0.479 0.0106 -0.278
1g (post) p.g/liter (post) 0.203 0.00222 0.0783
ng/mg of creatinine (pre) ng/mg of creatinine (pre) 0.175 -0.1 18 0.212
ng/mg of creatinine (post) ng/mg of creatinine (post) 0.61 1 -0.800 -0.228
270 Gonzalez-Ramirez at al. Vol. 272

TABLE 6
Changes in urinary porphyrin lsv&s among dental and nondental Personnel before and after DMPS challenge test
Each value is the mean ± S.E. The number of subjects used for the porphyrin determInations was Ieee than those used In the Hg studies because insufficient volumes
of urine were saved for that purpose.
Pentacarboxylporphyrin Precoproporphyrin Coproporphyrin
Group (n)
Before After Before After Before After
p4IlItSf

Dental Technicians (7) 2.1 ± 1.2 1.4 ± 0.9 1.0 ± 0.5 0.5 ± 0.5 38.5 ± 10.5 25.3 ± 9.7
Dentists (4) 2.5 ± 1 .3 2.3 ± 1 .4 1 .5 ± 0.4 0.3 ± 0.3 70.7 ± 25.5 47.8 ± 8.2*
NOndental (8) 3.2 ± I .0 1.9 ± 0.9 0.6 ± 0.3 0.6 ± 0.4 29.3 ± 8.0 14.0 ± 3.0
* Signfflcantty different from value before DMPS administratIon, P < .05.

TABLE 7
BehavIoral test results and P values based on the coefficient for urinary mercury level after DMPS with the regression analysis
controllIng for age, ethanol consumption, .ysgiass wear and vocabulary
Mean Values ± S.D. Unary Mercury
after OMPS
Unexposed Exposed P Value
Symbol-digit substitution response time (eec)
Unadjusted 19.7 ± 3.2 21 .2 ± 4.2
Adjusted 19.7 ± 2.3 21.2 ± 3.6 .00
The switching task (msec)
Unadjusted 11 755 ± 135 830 ± 266
Adjusted 1 1 756 ± 108 830 ± 253 .06
Unadjustedl2 678±136 817±258
Adjusted 12 678 ± 109 818 ± 252 .01
Unadjusted 13 626 ± 136 709 ± 236
Adjusted 13 626 ± I 18 709 ± 202 .04
Unadjusted 21 619 ± 141 704 ± 279
Adjusted 21 619 ± I 1 5 704 ± 241 .02
Unadjusted 22 636 ± 105 768 ± 263
22
Adjusted 636 ± 090 768 ± 226 .01
Unadjusted 23 531 ± 127 622 ± 222
Adjusted 23 531 ± 089 622 ± 205 .03
The one-hole test
Pin/mm
Unadjusted 38 ± 5 39 ± 5
Adjusted 38±3 39±3 a
Reach ± msec
Unadjusted 323 ± 55 320 ± 71
Adjusted 323 ± 38 328 ± 57 .02
Digit span
Unadjusted 5.9 ± .9 5.6 ± .8
Adjusted 5.9 ± .4 5.6 ± .6
sm- ±
Right
Unadjusted 41 1 ± 71 467 ± 81
Adjusted 41 1 ± 30 467 ± 77
Left
Unadjusted 414 ± 60 452 ± 84
Adjusted 414 ± 40 452 ± 21
a The exposed group performed better than the unexposed group.

their urinary mercury level only marginally higher than that It is of interest not only to compare the changes in the
of nonoccupationally exposed controls. This appears to be urinary mercury concentration after the DMPS challenge but
true in the United States, also, where a divided capsule, one also to compare the urinary mercury level ofthe three groups
part ofwhich contains elemental mercury and the other part before treatment (table 4). The differences between the non-
of which contains the alloy powder is commonly used to dental group and the dental technicians or dentists before
prepare amalgam. The capsule is shaken vigorously to break DMPS were highly significant. The difference between the
the divider and form the amalgam. Such a device and procedure dental technicians and dentist groups as far as -6- to 0-hr
for preparing amalgam is designed to minimize exposure of urinary Hg content (before administration) was not statisti-
dental personnel to mercury vapor but is not commonly used in cally significant (P = .252). After DMPS administration,
many developing countries for economic reasons. In Mexico, it is however, although the difference between the two dental
estimated that less than 1% of dentists use the amalgam cap- groups appeared marked, it was on the borderline of signif-
sules in their practice. In addition, Occupational Safety and icance (P = .057) This may be because of the small sample
Health Administration standards are not necessarily enforced size of the dentist group. The original expectation was that
in many developing countries. eight dentists from the clinic would participate in these ex-
1995 DMPS-M.rcUry Challenge Test 271

TABLE 8
Profile of mood scales and symptom P values based on urinary mercury level after DMP5 with the regression analysis controlling for
age, ethansi cons, eye wesr and vocabulary
Mean Values ± S.D. Urinary Mercury
- after DMPS
Unexposed Exposed P Value

Mood
Tension
Unadjusted 8.3 ± 3.6 9.8 ± 5.4
Adjusted 8.3 ± 1 .5 9.8 ± 4.4 .06
Depression
Unadjusted 4.1 ± 3.8 7.2 ± 10.2
Adjusted 4.1 ± 3.0 7.2 ± 7.2
Anger
Unadjusted 7.3 ± 3.1 9.7 ± 7.7
Adjusted 7.3 ± 2.6 9.7 ± 4.8 .05
Vigor
Unadjusted 16.9 ± 3.2 13.2 ± 4.5
Adjusted 16.9 ± 2.6 13.2 ± 2.3
Fatigue
Unadjusted 5.0 ± 4.2 4.6 ± 3.1
Adjusted 5.0 ± 2.1 4.6 ± 2.3
Confusion
Unadjusted 3.7 ± 2.0 5.5 ± 4.9
Adjusted 3.7 ± 1 .7 5.5 ± 3.7 .04
Symptoms
Headaches
Unadjusted 2.2 ± .4 2.5 ± .5
Adjusted 2.1 ± .3 2.5 ± .4 .00
Emotional
Unadjusted 4.2 ± .4 4.4 ± .5
Adjusted 4.2 ± .3 4.4 ± .3 .05
Coordination
Unadjusted 8.3 ± .6 8.3 ± .5
Adjusted 8.3 ± .5 8.3 ± .3
Comprehension
Unadjusted 3.1 ± .3 3.3 ± .5
Adjusted 3.1 ± .1 3.4 ± .3 .05
Memory
Unadjusted 4.4 ± .8 4.6 ± .9
Adjusted 4.4±.4 4.6±.7
Sensory
Unadjusted 6.7 ± .8 7.1 ± 1.2
Adjusted 6.7 ± .6 7.1 ± .8
Dizziness
Unadjusted 3.6 ± .9 4.0 ± 1.0
Adjusted 3.5 ± .1 4.0 ± .1
Poor sleep
Unadjusted 3.4 ± .9 3.8 ± 1.0
Adjusted 3.4±.5 3.8±.3
Skin disorders
Unadjusted 2.0 ± .0 2.2 ± .4
Adjusted 2.1 ± .0 2.2 ± .1
Chest
Unadjusted 4.1 ± .3 4.3 ± .6
Adjusted 4.1 ± .2 4.3 ± .4
Pain
Unadjusted 2.1 ± .3 2.7 ± 1.2
Adjusted 2.1 ± .1 2.7 ± .9
Odors
Unadjusted 1 .9 ± .3 2.3 ± 1.7
Adjusted 1.9 ± .2 2.3 ± .8
M symptoms
Unadjusted 2.8 ± 2.7 3.6 ± 3.0
Adjusted 2.8 ± 1.8 3.6 ± 1.4

periments. Because ofcircumstances beyond our control, only the lowest urinary mercury excretion before DMPS adminis-
five participated. tration. The working conditions and employment tasks of
The magnitude of the increased urinary excretion after these dental technicians have been described in the Study
DMPS was greatest in the case ofthe dental technician group Population section under Results to explain their mercury
(table 4). The greatest increase in each group after the DMPS exposure. Obviously, corrective measures should be taken in
challenge occurred in the individual in that group who had this clinic and perhaps in others in developing countries in
272 Gonzalez-Ramirez at al. Vol. 272

regard to better training in the handling ofmercury by dental ference, however, was found in the dentists. After the DMPS
technicians, better ventilation in the preparation room of the challenge, urinary porphyrin levels fell to 58% and 68% of the
dental clinic and the use of amalgam capsules. before DMPS values for dental technicians and nondental
It should not be overlooked that the DMPS challenge test personnel, respectively, which suggests depletion of renal
1) increased the mean urinary mercury excretion 35-, 49- and mercury.
88-fold in the respective study groups (table 4), 2) offered These findings are consistent with results ofprevious stud-
additional information on the body load of mercury because ies that demonstrate a highly significant dose and time-
there was no correlation between the before and after urinary related correlation between the urinary concentration of
mercury excretion ofthe dental technicians or controls (fig. 1) these three porphyrins and renal mercury content in rats
and 3) showed that the latter observation was not true for the (Woods et al., 1991) and also with observations that show a
dentists, perhaps because of the small number of dentists in significant association between urinary levels of these por-
the study. In addition, these results point out the tremendous phyrins and mercury exposure among dentists (Woods et al.,
capacity of the kidney for retaining mercury and the lack of 1993). The present results indicate the value of urinary por-
obvious clinical signs and symptoms ofmercury toxicity when phyrin measurements in addition to urinary mercury deter-
urinary mercury levels rise to 481, 275 and 37 gIliter of Hg minations for the assessment of the efficacy of DMPS in
in contrast to < 5 pg/liter ofHg, which is considered normal. reducing renal mercury levels.
Mercury levels in the urine of German dental personnel, Third, based on the results of neurobehavioral assays, the
before and after DMPS administration, have been reported present investigation found evidence of the occurrence of
by Zander et al. (1992). Dental nurses were reported to be subclinical effects caused by the exposure of humans to mer-
more exposed than dentists but this seemed to be related to cury, as indicated by urinary mercury concentrations that
the larger number of dental fillings that the dental nurses averaged between 30 and 481 pg/liter before and after the
had. In the present study, there was no linear correlation DMPS challenge. The pattern ofthe results ofthese neurobe-
between the urinary mercury level either before or after havioral tests provided particularly convincing evidence of
DMPS and the number of amalgams or the amalgam score the health effects at low levels of mercury exposure because
(data not shown). A possible reason for this is that the amal- they occurred in the younger, mercury-exposed group who do
gam restorations in the mouths of the subjects in this study not consume much alcohol compared with the reference
appear to have had much larger diameters than those found group of older subjects who did drink alcohol. Within the
in a previous study ofAmerican college students (Aposhian et constraints imposed by a small number of subjects, potential
al., 1992). cultural differences between the United States and Mexico
Second, urinary coproporphyrin concentration was found and relatively low exposure to mercury, the urinary mercury
to be well correlated with urinary total mercury amount or levels both before and after the DMPS challenge were ad-
concentration only in the case ofthe dental technicians when versely and statistically associated with functions associated
the pre-DMPS coproporphyrin was compared with the post- with complex attention (switching task), a psychomotor task
DMPS mercury (r = 0.921 for amount; r = 0.977 for concen- (symbol-digit latency), mood and symptoms in a linear dose-
tration; table 5). The pre-DMPS urinary coproporphyrin level effect manner. These results were consistent with previous
but not the post-DMPS urinary coproporphyrin levels ap- findings among dentists with low-level mercury exposure
pears to be indicative of mercury exposure. Based on the (Echeverria et al. , in press, 1995) and reflected similar sub-
evidence, the urinary mercury level after DMPS administra- clinical deficits found at higher exposures (Fawer et al. , 1983;
tion is a better indicator of the renal mercury burden than is Zedda et al. , 1980; Piikivi et al. , 1984; Soleo et al. , 1990;
the mercury concentration measured in the urine before Smith et al. , 1983) among other occupational populations.
DMPS was given. The implications of this for occupational The consistency in direction among most of the associations
medicine are obvious. The correlation between the pre-DMPS is noteworthy even though the differences in the results in
urinary coproporphyrin concentration and the post-DMPS the test for digit span and SRT did not meet statistical
urinary total mercury amount within these dentists (r = significance, perhaps because of the small number of sub-
0.667) was not as high as was found for the dental techni- jects. Improved performance among the exposed group for
cians. This may be due to the small size ofthe dentist group. the one-hole test may also be explained by the strong asso-
There was a negative correlation among the nondental group ciation with age, in which the younger group of subjects was
between the pre-DMPS urinary coproporphyrin concentra- expected to do better.
tion and post-DMPS urinary total mercury amount or con- A number of questions remain to be answered. First, does
centration (r = -0.642 amount; r = -0.716 concentration). DMPS given under the conditions of the challenge test mo-
This may be due to the fact that the nondental personnel did bilize mercury only from the kidney or does it also mobilize
not have a history ofrecent or extended exposure to mercury, mercury in other tissues and then allow the kidney to con-
as reflected by their low post-DMPS urinary mercury levels. centrate and excrete the mercury? Buchet and Lauwerys
Their coproporphyrin levels were lower than the other two (1989) reported that, when rats were given mercury vapor by
groups and may be in the normal range. inhalation, mercury accumulated mainly in the kidneys and
After the DMPS challenge, urinary porphyrin concentra- that DMPS mobilized the mercury stored in the kidney. After
tions declined with respect to pre-DMPS levels (table 6). The DMPS treatment, there was a decrease of the mercury con-
decrease in coproporphyrin level was most substantial in this tent of the kidney from about 30 g/100 g of body weight to
respect, decreasing to 68%, 66% and 48% ofthe before-DMPS about 4 g/100 g of body weight. Animal experiments, how-
values among dentists, dental technicians and nondental ever, often do not or sometimes cannot replicate environmen-
personnel, respectively, which suggests a substantial deple- tel conditions in humans. After a 4-day course of therapy
tion of renal mercury. The only statistically significant dif- with the chelating agent DMSA(2,3-dimercaptosuccinic acid)
1995 DMPS-Mercury Challenge Test 273

(which is
structurally analogous with DMPS) for workers References

exposed to large amounts of Hg#{176},

Bluhm et al. (1992) con- ALBERS, J. W., KALLENBACH, L. R., Fnx, L J., Lcoir, G. D., Wois, R. A.,
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