61, 224 –233 (2001)
Copyright © 2001 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
Effects of 2,3-Dimercapto-1-propanesulfonic Acid (DMPS) on Tissue
and Urine Mercury Levels following Prolonged
Methylmercury Exposure in Rats
Stephanie D. Pingree, P. Lynne Simmonds, and James S. Woods 1
Department of Environmental Health, University of Washington, 4225 Roosevelt Way NE, Suite 100, Seattle, Washington 98105
Received November 6, 2000; accepted January 30, 2001
Methylmercury, a potent neurotoxicant, accumulates in the
brain as well as the kidney during chronic exposure. We evaluated
the capacity of 2,3-dimercapto-1-propanesulfonic acid (DMPS), a
tissue-permeable metal chelator, to reduce brain, kidney, and
blood Hg levels and to promote Hg elimination in urine following
exposure of F-344 rats to methylmercury hydroxide (MMH) (10
ppm) in drinking water for up to 9 weeks. Inorganic (Hg 2ⴙ) and
organic (CH 3Hg ⴙ) mercury species in urine and tissues were assayed by cold vapor atomic fluorescence spectroscopy (CVAFS).
Following MMH treatment for 9 weeks, Hg 2ⴙ and CH 3Hg ⴙ concentrations were 0.28 and 4.80 ␮g/g in the brain and 51.5 and 42.2
␮g/g in the kidney, respectively. Twenty-four hours after ip administration of a single DMPS injection (100 mg/kg), kidney Hg 2ⴙ
and CH 3Hg ⴙ declined 38% and 59%, whereas brain mercury levels
were slightly increased, attributable entirely to the CH 3Hg ⴙ fraction. Concomitantly, Hg 2ⴙ and CH 3Hg ⴙ in urine increased by 7.2and 28.3-fold, respectively, compared with prechelation values. A
higher dose of DMPS (200 mg/kg) was no more effective than 100
mg/kg in promoting mercury excretion. In contrast, consecutive
DMPS injections (100 mg/kg) given at 72-h intervals significantly
decreased total mercury concentrations in kidney, brain, and
blood. However, the decrease in brain and blood mercury content
was restricted entirely to the CH 3Hg ⴙ fraction, consistent with the
slow dealkylation rate of MMH in these tissues. Mass balance
calculations showed that the total amount of mercury excreted in
the urine following successive DMPS injections corresponds quantitatively to the total amount of mercury removed from the kidney,
brain, and blood of MMH-exposed rats. These findings confirm the
efficacy of consecutive DMPS treatments in decreasing mercury
concentrations in target tissue and in reducing overall mercury
body burden. They demonstrate further that the capacity of DMPS
to deplete tissue Hg 2ⴙ is highly tissue-specific and reflects the
relative capacity of the tissue for methylmercury dealkylation. In
light of this observation, the inability of DMPS to reduce Hg 2ⴙ
levels in brain or blood may explain the inefficacy of DMPS and
similar chelating agents in the remediation of neurotoxicity associated with prolonged MMH exposure.
Key Words: 2,3-dimercapto-1-propanesulfonic acid (DMPS); chelation; mercury; methylmercury; mercuric ion; brain; blood; kidney.
1
To whom correspondence should be addressed. Fax: (206) 528-3550.
E-mail: [email protected].
Methylmercury, a potent neurotoxicant, readily penetrates
the blood-brain barrier through specific carrier-mediated transport systems (Miura et al., 1994). CNS damage by methylmercury results from preferential accumulation in focal parts of the
brain including the cerebellum and visual cortex, where it is
slowly dealkylated to Hg 2⫹ (Berlin, 1986). Degeneration and
atrophy of the sensory cerebral cortex, leading to paraesthesia,
ataxia, and hearing and visual impairment in the adult during
chronic methylmercury exposure have also been reported
(Miura et al., 1994). Prenatal exposure can cause cerebral
palsy, psychomotor retardation, delayed development, and
cognition changes (Clarkson, 1997). During prolonged exposure, methylmercury may also accumulate in the kidney and
promote renal toxicity (Woods et al., 1991).
A specific concern associated with methylmercury exposure
in humans is the need for effective therapy in dealing with
intoxication. In this respect, chelation therapy is the most
commonly used and seen as the least invasive (Aposhian,
1983). Chelating agents compete with the in vivo binding site
for the metal ion through the process of ligand exchange
(Jones, 1994). The toxic metal bound to the chelating agent is
excreted from the body through the urine or feces. Among
chelating agents currently available, the sodium salt of 2,3dimercapto-1-propane-sulfonate (DMPS) has been found to be
highly effective, particularly with respect to promoting mercury elimination following inorganic or elemental mercury
exposure (Aaseth et al., 1995; Aposhian 1983; Garza-Ocanas
et al., 1997; Gonzalez-Ramirez et al., 1995; Maiorino et al.,
1996). Remaining to be confirmed is the efficacy of DMPS in
reduction of mercury body burden resulting from organic mercury exposure, and in particular, its value in the removal of
inorganic Hg (Hg 2⫹) arising from dealkylation of methylmercury in target tissues.
In the present studies, we evaluated the efficacy of DMPS in
reducing Hg 2⫹ and CH 3Hg ⫹ from kidney, brain, and blood
mercury levels following prolonged methylmercury exposure
in rats. As part of this assessment, we performed mass balance
calculations to demonstrate the quantitative relationship between mercury removed from each tissue and that excreted in
the urine. We also employed linear regression analysis to
224
DMPS EFFECTS IN METHYLMERCURY-EXPOSED RATS
assess the strength of association between tissue mercury concentrations and urinary mercury levels in relation to DMPS
treatments.
MATERIALS AND METHODS
Materials. Male Fischer-344 rats (200 –225 g) were purchased from Simonsen Labs (Gilroy, CA). Methylmercury (II) hydroxide (CH 3HgOH, MMH)
was purchased from Alfa Aesar (Ward Hill, MA). 2,3-Dimercapto-1-propanesulfonic acid (DMPS) was obtained from Sigma Chemical Co. (St. Louis,
MO). All other chemicals were reagent grade and were purchased from
standard commercial sources. Distilled deionized water was used for preparation of all aqueous solutions.
Animal treatment. Immediately upon receipt, all animals were placed in
individual, wire bottom, hanging cages and given deionized water and food
(Wayne Rodent Blox rat chow) ad libitum. This diet has been shown to be free
of mercury content. Lighting was set at a 12-h light/dark cycle, and the
temperature of the animal housing facility was kept at 20°C. Following 1-week
acclimation, the animals were separated into 2 groups (described below) and
given either deionized water or drinking water containing 10 ppm MMH. This
dose of MMH was selected from previous studies (Fowler and Woods, 1977;
Woods and Fowler, 1977) as sufficient to permit significant target tissue
accumulation without evidence of overt neurotoxicity or renal injury during the
course of the treatment regimen.
Study design. Two prolonged methylmercury-exposure studies were undertaken. Study 1 was designed to evaluate the efficacy of DMPS dosage on
mercury clearance from brain and kidney. In this study, 30 rats were divided
into 2 groups of 18 and 12 each. The larger group of 18 animals was placed on
a continuous regimen of drinking water containing 10 ppm MMH, whereas the
remaining 12 animals were placed on deionized water (dH 2O) as controls.
After 9 weeks, all 30 rats were transferred to individual metabolism cages for
24-h urine collections. Immediately thereafter, the 18 animals that had received
MMH for 9 weeks were divided randomly into 3 groups of 6 each. The first 2
groups of 6 animals received single ip injections of DMPS at either 100 mg/kg
or 200 mg/kg, respectively, whereas the third group received saline injections.
Concurrently, the 12 control rats that had been maintained for 9 weeks on
dH 2O were divided into 3 groups of 4 each and given comparable injections of
100 or 200 mg/kg DMPS or saline. Subsequently, all rats were returned to
individual metabolism cages for post-treatment 24-h urine collections. All
animals were then sacrificed, and tissues were collected for mercury assessments.
The second study (Study 2) was designed to assess the effect of consecutive
DMPS treatments on mercury clearance from brain, kidney, and blood of
MMH-exposed rats. In this study, 30 rats were placed on a continuous regimen
of drinking water containing 10 ppm MMH for 6 weeks. The shorter MMH
exposure time in Study 2 was selected based on the observation from Study 1
that the equilibrium between organic and inorganic mercury species in kidney
cortex is achieved by 6 weeks of MMH exposure (described in Results).
Hence, the prechelation concentrations of both Hg 2⫹ and CH 3Hg ⫹ in renal
cortex are sufficient following 6 weeks of MMH exposure to permit evaluation
the efficacy of DMPS chelation in clearing both organic and inorganic mercury
species from kidney, as well as from brain and blood.
To determine the effects of multiple DMPS treatments on tissue mercury
levels, animals were given up to 3 DMPS injections over a period of 3–7 days
prior to sacrifice. For this study, the 30 MMH-exposed rats were divided into
2 groups of 18 and 12 animals, respectively. The first group of 18 rats received
a single ip injection of 100 mg/kg DMPS, whereas the remaining 12 were
given a saline injection, as controls. All 30 animals were then transferred to
individual metabolism cages for 24-h urine collections. Rats were denied food
but were provided dH 2O ad libitum during the urine collection period. Following urine collections, 6 animals from the DMPS-treatment group and 4 rats
from the control group were sacrificed, and brains, kidneys, and blood were
retrieved for mercury analyses. Seventy-two hours after the first injection, the
225
remaining 12 DMPS-treated rats were given a second 100mg/kg DMPS
injection, while the remaining 8 saline-treated rats were given a second saline
injection. After 24 h, 6 of the DMPS-treated rats and 4 of the control rats were
sacrificed and tissues collected. Seventy-two h after the second injection the
remaining animals were given a third DMPS or saline treatment. Twenty-four
h after the third injection all remaining rats were sacrificed and tissues
collected. Between DMPS treatments, animals were held in metabolism cages
without food but with dH 2O for 24-h urine collections and then returned to
their hanging cages and permitted food and water ad libitum for 48 hours. No
animals were deprived of food for more than 24 hours during the treatment
period. In all studies, animals were anesthetized by carbon dioxide (CO 2) and
then sacrificed by decapitation. Blood was obtained by cardiac puncture into
heparinized tubes prior to sacrifice. Brains and kidneys were harvested surgically immediately following sacrifice. All tissues were preserved at – 80°C
until mercury analysis.
Collection of urine samples. Animals were placed in hanging metabolism
cages for 24 h with free access to drinking water (containing either MMH or
dH 2O) but not food. The metabolism cages were fitted with metal funnels
attached to the bottom with a plastic ping-pong ball placed at the hole of the
funnel to allow urine but not feces to pass through. Aluminum foil-covered,
polypropylene 125-ml volumetric flasks were placed under the funnels to
collect the urine without allowing evaporation. At the end of 24 h the urine
volume was measured. The urine was then acidified with a drop of 6 N HCl and
frozen at –20°C until mercury analysis.
Mercury determination. Urinary mercury was measured using a modified
version of the digestion method by Corns et al. (1994). 2.5 ml of HCl and 2-ml
bromate/bromide solution was added to 2.5-ml urine sample and allowed to sit
overnight in a 20-ml glass scillation vial. Hydroxylammonium chloride (20%)
was then added to decolorize the sample and to stop the digestion process. The
fully digested sample was transferred to a 50-ml borosilicate glass volumetric
flask, and distilled water was added to a total volume of 50 ml. The total
(organic and inorganic) mercury content of the sample was then analyzed by
cold vapor atomic fluorescence spectroscopy (CVAFS) using a PSA Merlin
Mercury Analysis System (Questron Corp., Mercerville, NJ). For inorganic
mercury (Hg 2⫹) determinations, a 0.5-ml sample of urine was digested overnight in 2 ml of HNO 3. The sample was then diluted to a volume of 20 ml with
dH 2O, and the total Hg 2⫹ content was measured using CVAFS. The organic
mercury content of the sample was determined by the difference in the total
and inorganic mercury values.
The total and inorganic mercury concentrations in kidney and brain tissues
were analyzed by CVAFS following digestion and preparation of tissues as
described by Atallah and Kalman (1993). The organic mercury content of
tissue samples was again determined by the difference in the total and inorganic mercury concentrations.
Total and organic mercury concentrations in blood samples were directly
measured by ethylation-GC-CVAFS after alkaline digestion-solvent extraction, as described by Liang et al., 2000. Inorganic mercury was calculated as
the difference between total and organic mercury.
For each of the above cited procedures, validation of Hg 2⫹ and CH 3Hg ⫹
analysis was confirmed by concomitant measurement of control urine or tissue
samples containing a range of known concentrations of Hg 2⫹, CH 3Hg ⫹ or total
mercury (Hg 2⫹ ⫹ CH 3Hg ⫹) derived from standard reference materials. Mean
recoveries of Hg 2⫹ and CH 3Hg ⫹ from spiked tissues were on the order of
95–100% with no detectable cross contamination of mercury species, consistent with findings reported by original authors. All procedures employed
specific quality control protocols, including pre-analysis of all reagents and
materials used in the analyses, precluding potential Hg 2⫹ or CH 3Hg ⫹ contamination from sources other than biological samples under evaluation.
Statistical analyses. Data are presented as means ⫾ standard error of the
mean (SEM). Statistical analyses were conducted using Student’s t-test with
1-tailed distribution. p values less than 0.05 were considered significant.
Correlational analysis was performed using the Excel function (Microsoft,
Redmond, WA) and expressed as the correlation coefficient.
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PINGREE, SIMMONDS, AND WOODS
TABLE 1
Mercury Concentrations in Urine (␮g/ml) before and after DMPS Treatment
Exposure
CH 3Hg ⫹
Hg 2⫹
Total Hg
Treatment
Before
After
Before
After
Before
After
MMH
Saline
MMH
DMPS
100 mg/kg
DMPS
200 mg/kg
Saline
2.32
(0.13)
2.40
(0.12)
2.16
(0.08)
0.00
(0.00)
0.00
(0.00)
0.00
(0.00)
3.20
(0.12)
43.10*
(1.49)
43.44*
(0.82)
0.00
(0.00)
0.04
(0.01)
0.04
(0.00)
1.04
(0.06)
1.19
(0.06)
1.17
(0.04)
0.00
(0.00)
0.00
(0.00)
0.00
(0.00)
0.91
(0.03)
8.60*
(0.36)
8.63*
(0.34)
0.00
(0.00)
0.02
(0.01)
0.02
(0.00)
1.28
(0.11)
1.22
(0.08)
0.99
(0.04)
0.00
(0.00)
0.00
(0.00)
0.00
(0.00)
2.29
(0.10)
34.49*
(1.15)
34.81*
(0.60)
0.00
(0.00)
0.02*
(0.00)
0.02*
(0.00)
MMH
dH 2O
dH 2O
dH 2O
DMPS
100 mg/kg
DMPS
200 mg/kg
Note. The mean total, inorganic, and organic mercury concentrations (⫾ SEM) in the urine before and after DMPS treatment are presented for each exposure
group of Study 1. Rats were exposed to either MMH or to dH 2O for 9 weeks, then transferred to metabolism cages for 24-h urine collections, as described in
Materials and Methods. Thereafter, rats were given a single ip injection of DMPS (100 or 200 mg/kg) or saline and then returned to metabolism cages for
post-DMPS 24-h urine collections. Animals were then sacrificed and tissues were collected for mercury measurements. *Significantly different from the
saline-injected group (p ⱖ 0.05).
RESULTS
DMPS Promotes Urinary Excretion of Both Organic and
Inorganic Mercury Species
In Study 1 we evaluated the effects of DMPS dosage on
kidney and brain mercury levels and corresponding urinary
excretion of inorganic and organic mercury species following
prolonged MMH exposure in rats. As shown in Table 1,
inorganic and organic mercury concentrations in the urine of
the MMH-exposed animals before DMPS treatment were approximately equivalent, consistent with previous observations
(Woods et al., 1991). Following treatment with DMPS at either
100 or 200 mg/kg ip, urinary concentrations of both inorganic
and organic mercury species increased significantly, compared
to those of MMH-treated rats given a saline injection. The
inorganic and organic mercury concentrations in urine of the
animals given 100 mg/kg DMPS increased 9.5- and 15.1-fold,
respectively, compared with those given saline injections. Notably, however, administration of 200 mg/kg DMPS did not
promote a further increase in the concentration of either inorganic or organic mercury excreted in the urine, compared to
that observed following treatment with 100 mg/kg DMPS.
Also of interest is the observation that DMPS treatment at
either the 100 or 200 mg/kg dose level resulted in a detectable
increase in both inorganic and organic mercury concentrations
in the urine of rats exposed only to dH 2O, compared to those
receiving a saline injection. Organic mercury levels were significantly increased in urine of dH 2O-exposed rats following
DMPS treatment, suggesting the efficacy of DMPS in the
clearance of even very low levels of mercury derived from
ambient exposures.
A Single Dose of DMPS Reduces Kidney but Not Brain
Mercury Concentrations
Additional studies were undertaken to determine the corresponding effects of DMPS dosage on kidney and brain mercury
levels. In previous studies (Woods et al., 1991) we have
demonstrated that mercury accumulates in the kidney as well
as the brain during prolonged MMH exposure. Moreover,
methylmercury is demethylated to inorganic mercury (Hg 2⫹) in
the renal cortex, such that steady state equilibrium of organic
and inorganic mercury species is sustained over a wide range
of total mercury concentrations. Studies described here confirm
this finding. As shown in Table 2, the mean concentrations of
Hg 2⫹ and CH 3Hg ⫹ were 51.5 and 42.2 ␮g/g of renal cortex,
respectively, in saline-injected rats, representative of prechelation kidney mercury concentrations following MMH exposure
for 9 weeks. Treatment of MMH-exposed rats with DMPS at
100 mg/kg decreased both inorganic and organic mercury
concentrations to 37.2 ␮g/g (73%) and 26.5 ␮g/g (63%) of
prechelation levels, respectively. Treatment with DMPS at the
higher dose of 200 mg/kg produced a somewhat greater reduction in the kidney concentrations of both inorganic and organic
mercury species, to 33.4 ␮g/g (65%) and 18.9 ␮g/g (45%) of
prechelation values, respectively. However, differences in effects of treatment at 100 versus 200 mg/kg were not statistically significant.
Unlike as observed with the kidney, treatment of 9-week
MMH-exposed rats with a single dose of DMPS at either 100
mg/kg or 200 mg/kg was largely ineffective in reducing brain
mercury levels. As shown in Table 2, the mean concentrations
of Hg 2⫹ and CH 3Hg ⫹ were 0.28 and 4.8 ␮g/g of whole brain,
DMPS EFFECTS IN METHYLMERCURY-EXPOSED RATS
TABLE 2
Mercury Concentrations in Kidney and Brain
following DMPS Treatment
Kidney (␮g/g)
Exposure
Treatment
MMH
Saline
MMH
DMPS
100 mg/kg
DMPS
200 mg/kg
Saline
MMH
dH 2O
dH 2O
dH 2O
DMPS
100 mg/kg
DMPS
200 mg/kg
Brain (␮g/g)
Hg 2⫹
CH 3Hg ⫹
Hg 2⫹
CH 3Hg ⫹
51.48
(0.94)
37.24*
(1.28)
33.40*
(1.35)
0.09
(0.00)
0.03*
(0.00)
0.02*
(0.00)
42.19
(0.50)
26.52*
(0.56)
18.88*
(0.93)
0.02
(0.00)
0.03
(0.01)
0.02
(0.00)
0.278
(0.01)
0.360*
(0.01)
0.334
(0.01)
0.002
(0.00)
0.003
(0.00)
0.003
(0.00)
4.797
(0.02)
5.874
(0.17)
4.723
(0.14)
0.007
(0.00)
0.007
(0.00)
0.005
(0.00)
Note. The mean mercury concentrations (⫾ SEM) in the kidney and brain
following 9 weeks of MMH exposure and after DMPS or saline injection are
presented. Exposure and treatment regimens are as described in Table 1. The
mercury levels are shown for each exposure and treatment group. *Significantly different from the saline-injected group (p ⱖ 0.05).
respectively, in saline-injected rats, representative of prechelation brain mercury concentrations following 9 weeks of MMH
exposure. Of note, mercury in the brain was predominantly
(95%) CH 3Hg ⫹, indicative of the relatively slow dealkylation
of MMH to Hg 2⫹ in neuronal tissues (Berlin, 1986). Treatment
227
of rats with a single dose of DMPS at 100 mg/kg appeared to
slightly increase the brain concentrations of both organic and
inorganic mercury constituents. However, the inorganic and
organic mercury concentrations in brains of rats given a 200
mg/kg DMPS injection were not significantly different from
concentrations found prior to chelation.
Consecutive DMPS Injections Promote Successive
Urinary Elimination of Both Organic and
Inorganic Mercury Species
We conducted Study 2 to assess the effects of consecutive
DMPS treatments on mercury clearance from kidney, brain and
blood of MMH-exposed rats and to measure the corresponding
changes in urinary mercury levels. In these studies 30 rats were
exposed to MMH in drinking water for 6 weeks, followed by
1, 2, or 3 consecutive DMPS treatments (100 mg/kg, ip) at 72-h
intervals, as described in Materials and Methods. As shown in
Figure 1, consecutive DMPS treatments were highly effective
in promoting successive release of both organic and inorganic
forms of mercury into the urine. The inorganic and organic
mercury concentrations in the urine of MMH-exposed animals
prior to DMPS injection were 1.13 and 1.17 ␮g/ml, respectively, compared with 0.01 and 0.00 ␮g/ml, respectively, in
rats exposed only to dH 2O. Following a single DMPS injection, the total mercury concentration in 24-h urine samples
increased 18-fold, with 6- and 12-fold increases in inorganic
and organic species, respectively, compared with that observed
in urine of MMH-exposed rats receiving a saline injection. A
FIG. 1. Urine mercury concentrations following consecutive DMPS injections in MMH-exposed rats. The mean total, inorganic, and organic mercury
concentrations (⫾ SEM) excreted in 24-h urine samples of MMH-exposed rats following up to 3 consecutive DMPS or saline injections are shown. DMPS (100
mg/kg) or saline were administered by ip injection at 72-h intervals according to the regimen described under Materials and Methods. The initial DMPS injection
was given immediately following cessation of MMH exposure (10 ppm in drinking water for 6 weeks). Asterisks (*) indicate significantly different (p ⬍ 0.05)
from saline-injected (prechelation) value. In this and other figures, n ⫽ 6 for DMPS-injected and n ⫽ 4 for saline-injected.
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PINGREE, SIMMONDS, AND WOODS
FIG. 2. Kidney mercury concentrations following consecutive DMPS injections in MMH-exposed rats. The mean total, inorganic, and organic mercury
concentrations (⫾ SEM) found in kidney cortex of MMH-exposed rats following up to 3 consecutive DMPS injections are shown. DMPS (100 mg/kg) or saline
were administered by ip injection at 72-h intervals according to the regimen described under Materials and Methods. The initial DMPS injection was given
immediately following cessation of MMH exposure (10 ppm in drinking water for 6 weeks). Asterisks (*) indicate significantly different (p ⬍ 0.05) from
saline-injected (prechelation) value.
second DMPS injection administered 72 h following the first
was equally effectively in promoting mercury excretion, the
total urinary mercury concentration increasing to 27 times that
observed in MMH-exposed animals receiving 2 consecutive
saline injections. A third DMPS injection given 72 h after the
second promoted additional excretion of both organic and
inorganic mercury species, the total urinary mercury concentration increasing another 18-fold that seen in urine of salineinjected controls. Quantitatively, mean total urine mercury
concentrations following the first, second, and third DMPS
injections were 24.8, 25.6, and 14.4 ␮g/ml, respectively. The
sum of total mercury excreted in the urine following all 3
DMPS injections was 64.8 ␮g/ml, compared with 4.4 ␮g/ml
after 3 consecutive saline injections. Creatinine adjustment of
urinary mercury concentrations showed a slightly more pronounced mercury excretion trend associated with the number
of DMPS injections when compared with that of the noncreatinine adjusted results, although no trend in creatinine excretion in the urine following DMPS administration was seen (not
shown). DMPS treatments did not affect mean total urine 24-h
excretion rates, which remained constant (12.2 ⫾ 1.3 ml/24 h)
throughout the treatment regimen.
Consecutive DMPS Treatments Were Effective in Promoting
Release of Mercury from the Kidney, Brain and
Blood of MMH-Exposed Rats
Figure 2 represents changes in kidney mercury concentrations following each of the 3 consecutive DMPS injections.
Prior to chelation, the total mercury concentration found in
kidneys of MMH-exposed rats given a saline injection was
75.6 ⫾ 5.7 ␮g/g. The kidney total mercury concentration
decreased significantly following each DMPS treatment. A
single DMPS injection produced a decrease in total kidney
mercury content to 55.2 ␮g/g, approximately 73% of that
found in kidneys of saline-treated controls. Successive DMPS
treatments produced additional reductions in total kidney mercury content to 59 and 55% of remaining control levels, respectively. Organic and inorganic mercury constituents appeared to be cleared to a similar extent following consecutive
DMPS treatments, suggesting comparable availability of Hg 2⫹
and CH 3Hg ⫹ for ligand exchange with thiol groups of DMPS
in kidney cells. Notably, a slight (but not significant) increase
in both inorganic and organic mercury levels was seen in
kidneys of MMH-exposed animals following the first and
second saline injections (stippled bars in Fig. 2). This trend
possibly represents continued partitioning of mercury from
blood to kidneys following cessation of mercury exposure.
The effects of consecutive DMPS treatments (100 mg/kg) on
brain mercury levels are described in Figure 3. Similar to
observations made in Study 1 (Table 2), brain total mercury
content increased slightly within the 24-h period following a
single DMPS injection. This increase appeared to be attributable principally to the organic mercury fraction. However, as
shown in Figure 3, brain mercury levels decreased following
subsequent DMPS treatments, the most notable decrease occurring after the third DMPS injection. Both total mercury and
the organic mercury fraction were decreased to approximately
60% of levels found in brains of MMH-exposed rats receiving
DMPS EFFECTS IN METHYLMERCURY-EXPOSED RATS
229
FIG. 3. Brain mercury concentrations following consecutive DMPS injections in MMH-exposed rats. The mean total, inorganic, and organic mercury
concentrations (⫾ SEM) found in whole brains of MMH-exposed rats following up to 3 consecutive DMPS injections are shown. DMPS (100 mg/kg) or saline
were administered by ip injection at 72-h intervals according to the regimen described under Materials and Methods. The initial DMPS injection was given
immediately following cessation of MMH exposure (10 ppm in drinking water for 6 weeks). Asterisks (*) indicate significantly different (p ⬍ 0.05) from
saline-injected (prechelation) value.
3 consecutive saline injections. The inorganic mercury concentration remained largely unchanged with successive DMPS
treatments. As noted previously, however, inorganic mercury
comprises less than 10% of total mercury found in the brain of
rats following prolonged MMH exposure.
Further studies were conducted to evaluate the effects of
consecutive DMPS injections on blood mercury levels. As
shown in Figure 4, mercury in the blood of MMH-exposed rats
is present principally as the organic form, comparable to that
observed in the brain. DMPS was highly effective in reducing
FIG. 4. Blood mercury concentrations following consecutive DMPS injections in MMH-exposed rats. The mean total, inorganic, and organic mercury
concentrations (⫾ SEM) found in whole blood (wet basis) of MMH-exposed rats following up to 3 consecutive DMPS injections are shown. DMPS (100 mg/kg)
or saline were administered by ip injection at 72-h intervals according to the regimen described under Materials and Methods. The initial DMPS injection was
given immediately following cessation of MMH exposure (10 ppm in drinking water for 6 weeks). Asterisks (*) indicate significantly different (p ⬍ 0.05) from
saline-injected (prechelation) value.
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PINGREE, SIMMONDS, AND WOODS
TABLE 3
Mass Balance Analysis of Mercury following Consecutive DMPS Injections
Mercury change following DMPS injection
Treatment
Urine (␮g)
⌬Kidney (␮g)
⌬Brain (␮g)
⌬Blood (␮g)
Urine-store
First DMPS injection
Second DMPS injection
Third DMPS injection
274.3
623.1
794.1
–39.0
–62.8
–74.8
0.6
–0.4
–2.0
–324.0
–442.8
–445.7
88.1
117.1
–241.6
Note. Rats were exposed to MMH (10 ppm.) for 6 weeks in drinking water, then treated with up to 3 consecutive injections of DMPS (100 mg/kg) at 72-h
intervals. Tissues and urine were harvested and mercury content was measured as described in Materials and Methods. Values represent the cumulative total
amount (␮g) of Hg excreted in the urine following each consecutive DMPS treatment, compared with the concomitant change in mercury content in kidney, brain,
or blood. Urine-store represents the difference in total mercury found in the urine versus the sum total amount of mercury eliminated from tissues.
blood mercury levels. As with the brain, the decrease in blood
mercury was attributable principally to the organic mercury
content. A single DMPS injection reduced the blood total
mercury concentration to73% of that found in blood of MMHexposed rats given a saline injection. Second and third consecutive DMPS treatments reduced the total blood mercury content by an additional 50 and 40% of the remaining blood
mercury levels, respectively. Inorganic mercury concentrations
in both the saline- and DMPS-injected animals appeared to
increase slightly with the first and second consecutive DMPS
injections and to decline after the third. However, these
changes were not statistically significant. Second and third
DMPS injections decreased the organic mercury fraction in
blood to 53 and 61%, respectively, of that in blood of MMHexposed rats given a saline injection.
The Increase in Urinary Mercury Corresponds
Quantitatively to Reduction in Tissue Mercury
Concentrations following DMPS Treatment
Mass balance analysis was performed to calculate the
amount of total mercury removed from kidney, brain and blood
following each DMPS injection compared with the amount of
total mercury eliminated in the urine. The amount of mercury
eliminated following each DMPS treatment was calculated in
␮g of mercury excreted in 24 h of urine collection after
injection. Computations are presented in Table 3. Following
consecutive DMPS treatments, the average concentration of
mercury excreted after the previous injection was added to the
amount found in the present 24-h collection to determine the
total cumulative amount of mercury excreted following each
consecutive injection. The mercury eliminated from each tissue
was calculated by taking the average mercury concentration
found in the tissue of the saline-treated animals (representing
the prechelation mercury content), and subtracting the average
mercury concentration found in the tissue following DMPS
treatment. This value was then multiplied by the mean mass of
the tissue for kidney (2.0 g) and brain (1.8 g), or the volume of
the blood of the average adult male rat (13.5 ml) (Davies and
Morris, 1993). This value represents the total amount of mer-
cury that was eliminated from the tissue following DMPS
injections. As shown in Table 3, the amount of mercury excreted in the urine following the first DMPS injection accounted for approximately 76% of the total amount of mercury
eliminated from kidney, brain, and blood. The amount of
mercury excreted in the urine following the second and third
DMPS injections, however, exceeded the amount of mercury
eliminated from kidney, blood, and brain, possibly reflecting
depletion of mercury from other body stores (e.g., the hepatobiliary system or fatty tissues) with consecutive DMPS treatments.
Urinary Mercury Content following Successive DMPS
Treatments Corresponds to Mercury Body Burden
Finally, linear regression analysis was performed and correlation coefficients (r) were calculated to assess the strength of
the association between tissue mercury body burden and urinary mercury concentrations before and after successive
DMPS treatments. Computations are presented in Table 4.
Tissue mercury levels measured in saline-injected rats were
employed as prechelation mercury levels. For these assessments, mercury body burden was defined as the amount of total
mercury removed from (1) the kidney, (2) the kidney and brain
combined, and (3) the kidney, brain and blood combined
(prechelation – postchelation values) after 6 weeks of MMH
exposure (Study 2). Mercury excretion following consecutive
DMPS injections was calculated by adding the average total
24-h mercury concentration in the urine after the first, second,
or third consecutive DMPS injection. As seen in Table 4, all 3
measures of mercury body burden were found to be strongly
inversely correlated with the amount of mercury excreted in the
urine following the first DMPS injection (r ⬃ – 0.65 to – 0.84),
as well as for kidney and kidney plus brain following the
second DMPS treatment (r ⬃ – 0.79 to – 0.85). Weaker associations were found with all 3 measures, however, following
the third DMPS treatment. The correlation was inversely proportional, indicating that, as the mercury excreted in the urine
increases, the mercury present in tissues decreases. Notably,
these strong correlations were retained for all 3 measures of
DMPS EFFECTS IN METHYLMERCURY-EXPOSED RATS
TABLE 4
Linear Association of Hg Body Burden
with Urinary Hg Concentration
Correlation coefficient (r)
Injection
Kidney
Saline
First DMPS
Second DMPS
Third DMPS
Kidney and brain
Saline
First DMPS
Second DMPS
Third DMPS
Kidney, brain, blood
Saline
First DMPS
Second DMPS
Third DMPS
Total Hg
Hg 2⫹
CH 3Hg ⫹
–0.236
–0.654
–0.854
–0.053
–0.318
–0.549
–0.675
–0.604
0.038
–0.770
–0.473
0.601
–0.240
–0.664
–0.794
0.183
–0.316
–0.514
–0.666
–0.598
0.025
–0.814
–0.393
0.734
0.676
–0.839
–0.351
0.966
–0.114
–0.452
0.078
–0.232
0.643
–0.807
–0.434
0.964
Note. Correlation coefficients (r) for the linear association of mercury body
burden with inorganic mercury excretion in the urine following a first, second,
or third DMPS injection or saline treatment are shown. Negative values
indicate inverse correlations.
body burden when evaluated in relation to CH 3Hg ⫹ alone,
consistent with the ability of DMPS to effectively reduce the
organic mercury concentration of all 3 tissues. In contrast, the
addition of brain and blood to the kidney as measures of body
burden resulted in weaker associations for total mercury and
Hg 2⫹, reflecting the lack of effect of DMPS on Hg 2⫹ in these
tissues. As expected, the correlation between mercury body
burden and prechelation urinary mercury excretion (i.e., salineinjected) was weak in each case. These findings support the
established view that the post-DMPS chelation urinary mercury concentration is more representative of mercury body
burden than prechelation levels (Aposhian et al., 1995; Echeverria et al., 1998).
DISCUSSION
Numerous studies have documented the efficacy of DMPS in
promoting the elimination of mercury and other heavy metals
in animals and human subjects (Aaseth et al., 1995; Aposhian
et al., 1992; Cherian et al., 1988; Clarkson et al., 1981; de la
Torre et al., 1996; Garza-Ocanas et al., 1997; Hurlbut et al.,
1994; Keith et al., 1997). Particular attention in this respect has
been paid to DMPS as an effective chelator of inorganic
mercury derived from exposure to elemental or inorganic mercury compounds. Few studies, however, have sought to determine the efficacy of DMPS in clearance of mercury species
from target tissues following prolonged exposure to organic
mercury compounds. The present studies demonstrate that
231
DMPS effectively clears both organic and inorganic mercury
species from target tissues of rats following exposure to MMH
for prolonged periods. However, the efficacy of DMPS in this
respect appears to reflect of the relative capacity of the specific
tissue for methylmercury dealkylation. Moreover, the efficacy
of DMPS as a mercury chelator appears to be related to
consecutive administration in consistent dosages, rather than to
the magnitude of the dose received.
The dissociation of methylmercury into organic and inorganic species in the kidney during prolonged exposure to
MMH in rats has been previously described (Woods et al.,
1991). Of note, an equilibrium between Hg 2⫹ and CH 3Hg ⫹
concentrations in the kidney cortex is readily achieved in a
dose- and time-dependent manner during the course of continuous MMH exposure, and this equilibrium is maintained by the
kidney over a wide range of total mercury concentrations to
approximately 100 ␮g/g cortex. Inasmuch as both Hg 2⫹ and
CH 3Hg ⫹ may participate in renal toxicity, it is notable that
DMPS is equally effective in depleting both species from
kidney tissue. Analysis of the data presented here shows a
direct correlation between the increases in the urinary concentrations of both organic and inorganic mercury fractions and
the decline in the concentrations of each species in kidney
following DMPS treatment. These findings are consistent with
the very high ligand exchange rates (10 9/s) of both Hg 2⫹ and
CH 3Hg ⫹ for thiol groups (Martin, 1986), and suggests comparable availability of organic and inorganic Hg species for
exchange with DMPS in kidney cells.
Consistent with the effects on renal mercury content, DMPS
effectively reduced blood total mercury concentrations over the
course of consecutive treatments. Unlike in the kidney, however, less than 5% of total mercury in the blood was present as
Hg 2⫹. The effect of DMPS in lowering total blood mercury was
restricted entirely to the organic mercury constituent, which
decreased significantly following the second and third consecutive DMPS injections. Previous studies (Planas-Bohne, 1981)
have demonstrated that DMPS is moderately effective in removing methylmercury from blood following administration to
rats. However, the effects of DMPS on the inorganic Hg
fraction derived from demethylation of methylmercury in
blood cells was not reported. This is the first report to our
knowledge to assign the effect of DMPS on blood mercury to
the organic fraction.
The dissociation of methylmercury into organic and inorganic species in the brain during prolonged exposure of rats to
MMH was also noted in the present study, although the formation of Hg 2⫹ from MMH was substantially lower than that
observed in kidney. In this respect, the changes in total brain
mercury content observed during the course of MMH exposure
appeared to largely reflect the CH 3Hg ⫹ constituent, similar to
that observed in blood. Berlin (1986) reported that approximately 5–10% of methylmercury is demethylated to Hg 2⫹ in
the brain, consistent with this observation. Of note, DMPS was
effective in decreasing total brain mercury content in MMH-
232
PINGREE, SIMMONDS, AND WOODS
exposed rats only following 3 consecutive treatments. Similar
findings have been reported with respect to elemental mercury
exposure (Cikrt et al., 1996). The initial increase in brain
mercury content seen following a single DMPS injection is of
interest, inasmuch as it suggests that DMPS may facilitate
redistribution of CH 3Hg ⫹ from blood to brain until blood
mercury levels are concomitantly depleted by subsequent
DMPS treatments, or until DMPS accumulates in sufficient
concentrations in the CNS to effect significant mercury chelation. Notably, the partitioning of DMPS into the CNS is
relatively slow due to its polarity and limited lipid solubility
(Jones, 1994), consistent with this idea. The failure of DMPS
to cause significant depletion of Hg 2⫹ from the brain may
reflect the slow rate of demethylation of methylmercury in the
CNS and consequent low concentrations of Hg 2 ⫹relative to
those of CH 3Hg ⫹ available for exchange with DMPS. Alternatively, the efficacy of DMPS in extracting CH 3Hg ⫹ may
reflect the greater availability of organic mercury following
distribution from the blood to the CNS, as compared with that
of Hg 2⫹, which most likely is formed following compartmentalization of methylmercury to specific regions of the CNS that
may be poorly accessible to DMPS. The latter possibility raises
concerns regarding the efficacy of DMPS or similar chelating
agents in the remediation of neurotoxicity associated with
methylmercury, since principal adverse effects may be attributable to Hg 2⫹ accumulation following partitioning of methylmercury to specific CNS foci during prolonged exposure
(Aschner and Aschner, 1990; Friberg and Mottet, 1989). In
contrast to the present findings, Aposhian et al. (1996) reported
that DMPS did not alter brain mercury levels in rats treated by
ip injection with HgCl 2. This distinction may reflect differences in the capacity of DMPS complexes of Hg 2⫹ versus those
of CH 3Hg ⫹ to partition from blood into the CNS.
Although total mercury tissue levels increased significantly
during the course of MMH exposure, the mercury concentration in the urine increased only slightly during this period
(Table 1 and Fig. 1). These findings are consistent with the
view that prechelation urinary excretion is not well correlated
with mercury body burden (Aposhian et al., 1995). In contrast,
urinary mercury levels subsequent to DMPS chelation have
been shown to constitute a better approximation of Hg body
burden, particularly in the case of elemental mercury exposure
(Echeverria et al., 1998). Mass balance calculations performed
in the present investigation extend these findings to organic
mercury exposure, since the amount of mercury excreted in the
urine following consecutive DMPS treatments accounted for
essentially all of the mercury found in blood and the 2 principal
target tissues, kidney, and brain. Notably, essentially all of the
inorganic mercury excreted postchelation was of renal origin,
since DMPS treatment did not significantly reduce Hg 2⫹ concentrations in either blood or brain. These findings support the
view that postchelation urinary mercury levels are an accurate
measure of mercury body burden attributable to prolonged
methylmercury exposure.
In conclusion, the present studies demonstrate the efficacy of
DMPS in depleting mercury body burden and confirm previous
observations regarding the utility of postchelation urinary mercury levels as an accurate measure of body burden attributable
to prolonged methylmercury exposure. The finding that DMPS
readily depletes both Hg 2⫹ and CH 3Hg ⫹ from kidney, but only
CH 3Hg ⫹ from brain and blood, suggests that the capacity of
DMPS to remove Hg species reflects the tissue-specific dealkylation rate of MMH and, hence, the relative amount of
species present. These findings may explain the relative inefficacy of DMPS and similar chelators in the remediation of
neurotoxicity associated with organic mercury exposure, in
which Hg 2⫹, a principal mediator of toxicity, is present only in
low concentrations.
ACKNOWLEDGMENTS
This research was supported in part by University of Washington Center
Grant P30 ES07033 and by University of Washington Superfund Program
Project Grant ES04696 from the National Institute of Environmental Health
Sciences, NIH. Additional support was provided by the Wallace Research
Foundation.
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