Toxicokinetics of mercuric chloride and methylmercuric chloride in mice.

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Toxicokinetics of mercuric chloride and methylmercuric chloride in mice Jesper Bo Nielsen

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Department of Environmental Medicine , Odense University , Winsl⊘wparken 17, Odense C, Denmark , DK‐5000 Published online: 20 Oct 2009.

To cite this article: Jesper Bo Nielsen (1992) Toxicokinetics of mercuric chloride and methylmercuric chloride in mice, Journal of Toxicology and Environmental Health: Current Issues, 37:1, 85-122, DOI: 10.1080/15287399209531659 To link to this article: http://dx.doi.org/10.1080/15287399209531659

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TOXICOKINETICS OF MERCURIC CHLORIDE AND METHYLMERCURIC CHLORIDE IN MICE Jesper Bo Nielsen

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Department of Environmental Medicine, Odense University, Denmark Future human exposure to inorganic mercury will probably lead to a few individuals occupationally exposed to high levels and much larger populations exposed to low or very low levels from dental fillings or from food items containing inorganic mercury; human exposure to methylmercury will be relatively low and depending on intake of marine food. Ideally, risk assessment is based on detailed knowledge of relations between external and internal dose, organ levels, and their relation to toxic symptoms. However, human data on these toxicokinetic parameters originate mainly from individuals or smaller populations accidentally exposed for shorter periods to relatively high mercury levels, but with unknown total body burden. Thus, assessment of risk associated with exposure to low levels of mercury will largely depend on data from animal experiments. Previous investigations of the toxicokinetics of mercuric compounds almost exclusively employed parenteral administration of relatively high doses of soluble mercuric salts. However, human exposure is primarily pulmonary or oral and at low doses. The present study validates an experimental model for investigating the toxicokinetics of orally administered mercuric chloride and methylmercuric chloride in mice. Major findings using this model are discussed in relation to previous knowledge. The toxicokinetics of inorganic mercury in mice depend on dose size, administration route, and sex, whereas the mouse strain used is less important. The "true absorption" of a single oral dose of HgCI2 was calculated to be about 20% at two different dose levels. Earlier studies that did not take into account the possible excretion of absorbed mercury and intestinal reabsorption during the experimental period report 7-10% intestinal uptake. The higher excretion rates observed after oral than after intraperitoneal administration of HgCI2 are most likely due to differences in disposition of systemically delivered and retained mercury. After methylmercury administration, mercury excretion followed first-order kinetics for 2 wk, independently of administration route, strain, or sex. However, during longer experimental periods, the increasing relative carcass retention (slower rate of excretion) caused the elimination to deviate from first-order kinetics. Extensive differences in the toxicokinetics of methylmercury with respect to excretion rates, organ deposition, and blood levels were observed between males and females. Despite increasing knowledge on the toxicokinetics of mercuric compounds, the experimental basis for a risk assessment in relation to human exposure could still be improved, and suggestions for further experimental studies are briefly discussed.

This study was supported by grant 12-8108 from the Danish Medical Research Council to Jesper Bo Nielsen. Requests for reprints should be sent to Jesper Bo Nielsen, Department of Environmental Medicine, Odense University, Winsløwparken 17, DK-5000 Odense C, Denmark.

85 Journal of Toxicology and Environmental Health, 37:85-122, 1992 Copyright © 1992 by Hemisphere Publishing Corporation

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INTRODUCTION Mercury is liberated from the earth's crust to the biosphere through degassing from volcanic areas or evaporation from the oceans. In this context, release of mercury from the midatlantic ridge reaching from the Azores in the south toward Iceland in the north is an important natural source in the northern hemisphere. Mercury is emitted in the form of elemental vapor (Hg°). The natural emissions are estimated to be between 2700 and 6000 tons per year (Lindberg et al., 1987). Human activities have been estimated to add another 2000-3000 tons to the total release of mercury to the environment (Lindberg et al., 1987). Part of the emitted inorganic mercury becomes oxidized to Hg2+ and then methylated or in other ways transformed into organomercurials. The methylation is believed to involve a nonenzymatic reaction between Hg2+ and a methyl cobalamine compound (analogue of vitamin B12) that is produced by bacteria (Wood and Wang, 1983). This reaction takes place primarily in aquatic systems. The intestinal bacteria flora of various animal species including fish are also, though to a much lower degree, able to convert ionic mercury into methylmercuric compounds. Despite the limited amount of inorganic mercury released from anthropogenic sources on a world-wide basis, the importance of this source as an environmental toxin is indisputable as the release is most often concentrated into confined industrial areas (e.g., Minamata Bay and the Agano River in Niigata, Japan). The use of inorganic mercuric compounds has decreased during recent years, and improved precautions against industrial emissions have decreased the number of people at risk for accidentally high exposure to inorganic mercury. However, metallic mercury is still used extensively for dental fillings (amalgam). Future human exposure to inorganic mercury will therefore probably lead to few cases of occupationally highly exposed people and larger populations exposed to low or very low levels from dental fillings or from food items containing mercury (approximately 20-40% of the mercury in fish is inorganic). Human nonaccidental exposure to organomercurials and especially methylmercury is almost exclusively caused by intake of fish and marine mammals. However, several thousand severe intoxications have occurred in Iraq, causing more than 450 deaths due to baking bread from methylmercury-treated grain (Bakir et al., 1973). As dietary methylmercury is almost 100% absorbed and excreted slowly, it is accumulated in the body, and animal species at the highest trophic level contain the highest concentration of mercury. Thus, concentrations of mercury in swordfish, tuna, seals, and whales are normally in the region of 500-1000 /ig Hg/kg, though the mean value of mercury in muscles of pilot whales around the Faroe Islands is 2200 jig Hg/kg (Andersen et al., 1987). The concentration of mercury in fish from unpolluted areas is normally in the

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range of 50-400 pg Hg/kg, depending on factors such as pH and redox potential of the water, species, and age and size of the fish (EHC 101, 1990). Furthermore, the general population is occasionally exposed to organomercurials through the use of different pharmaceuticals. Thus, tiomersal is used for preservation of vaccines and immunoglobulins (an amount of 100 ng tiomersal per injection, corresponding to a subcutaneous dose of 0.1 ^mol Hg/kg in a 5-kg child). Despite an increased awareness of the toxicity of mercuric compounds, the general population will still be exposed to mercury in the future. The human exposure (oral or pulmonary) will normally be quite low, and will depend to a major extent on the intake of marine food sources.


Human Toxicity of Mercuric Chloride

The acute and chronic toxicity of inorganic mercury in humans has been studied during several centuries (Paracelsus, 1567; Ramazzini, 1700). After acute oral human intoxication with inorganic mercury salts, the immediate critical organs are the epithelia of the gastrointestinal tract due to corrosive effects of mercuric salts toward the mucous membranes (Berlin, 1986). Depending on the size of the mercury dose, kidney damage may subsequently develop (Berlin, 1986; Ellenhorn and Barceleux, 1987). Human studies on the toxicokinetics of mercuric mercury primary investigated the body retention and the rate of elimination calculated from whole-body countings or from fecal and urinary excretion rates (Miettinen, 1973; Rahola et al., 1973). Rahola et al. (1973) observed threephase excretion kinetics after oral administration of mercuric mercury to humans. The most rapid phase with a half-time less than 2 d corresponded to fecal elimination of nonabsorbed mercury during the first 1 2 d after dosing. The slowest excretion phase indicated an apparent intestinal absorption of at least 7%. However, as part of the intestinally absorbed mercury is reexcreted to the intestinal tract during the initial experimental period, the experiment did not offer data on the intestinal absorption, but rather on the whole-body retention of mercury. Thus in metal uptake studies employing whole-body counting, a clear distinction must be made between whole-body retention and the intestinal absorption. Ideally, risk assessment would be based on detailed knowledge on the relation between external and internal dose, blood/urine levels, organ levels, and their relation to toxic symptoms. However, data on organ deposition and relative organ distribution of mercury in humans after exposure to mercuric mercury are scarce, and are mainly obtained from individuals accidentally exposed to high mercury levels but with unknown total body burden. Therefore, risk assessment associated with

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exposure to low levels of mercury is presently based on blood or hair levels in relation to frequency of symptoms. However, there is no knowledge on mercury concentrations in critical organs. Therefore, the relation between exposure indices and organ levels is solely based on animal experiments.


Toxicokinetics of Mercuric Mercury in Experimental Animals

Two days after subcutaneous administration of 2.5 /xmol/kg body weight (b.w.) HgCI2 to rats, the whole-body retention was 76% (Magos and Webb, 1976). In comparison, the whole-body retention in rats given 1.25 /*mol Hg(NO3)2/kg b.w. by intramuscular injection was 73% at 2 d after dosing (Rothstein and Hayes, 1960). In the same study, the residual body burden was about 40% of the dose 15 d after a single intramuscular or intravenous injection of Hg(NO3)2 corresponding to 50 fig of mercury per animal. The whole-body counting curves (both after im and iv administration) showed 2 distinct phases of excretion during the first 2 wk after dosing. The first phase was rapid (half time of a few days), involving elimination of approximately 35% of the injected dose. The second phase had a half-time of about 30 d (Rothstein and Hayes, 1960). After intravenous injection of 5-50 fig mercury as Hg(NO3)2 into rats, a dose dependency was not observed in whole-body retention. However, the number of animals per group was only three and an effect of dose on organ distribution was actually indicated (Rothstein and Hayes, 1960). The initial clearance rate after intraperitoneal injection of 1-100 ng Hg as Hg(NO3)2 to rats or HgCI2 to mice was dose dependent (Cember and Donagi, 1963; Kristensen and Hansen, 1980). However, with respect to the rat study (Cember and Donagi, 1963), the conclusion concerning effect of dose on excretion is based on a single measurement of the initial excretion rate of 203Hg (d 1), which was described as 106% of the administered dose. Thus, the present knowledge on the effect of dose on the excretion of inorganic mercury is scarce, and experimental conditions could have influenced the conflicting results available. Human Toxicity of Methylmercury

The toxicity of methylmercury is mainly due to effects on the nervous system. In humans, toxic effects have been reported against both the peripheral and the central nervous systems. Significant differences exist between the toxicity of methylmercury following adult and prenatal exposure. These differences are both qualitative and quantitative. Thus, the main toxic effect following exposure of adults is impairment of the sensory, the visual, and the auditory functions together with an effect on those areas of the brain involved in coordination, whereas the toxic effect in the fetus is directed more toward the general development of the



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nervous system. The fetus is regarded as more sensitive to methylmercury toxicity than adults. Analysis of frequencies of signs and symptoms in several thousand intoxicated inhabitants of villages in Northern Iraq that for a couple of months ate bread baked from grain treated with methylmercury indicate that the earliest effects are paresthesias, malaise, and blurred vision (Bakir et al., 1973). At higher exposure levels, symptoms such as ataxia, dysarthria, deafness, and eventually death will occur (Bakir et al., 1973). The World Health Organization (WHO) recently estimated that a level of mercury in blood of 200 jtg/L, which at the exposure conditions during the Iraq intoxication episode corresponds to about 50 jug/g in hair, would cause an approximately 5% increase in the incidence of paresthesia (EHC 101,1990). The general population with a much lower level of mercury in blood would not be expected to experience toxic effects caused by methylmercury, though certain population groups with very high consumption of marine food may reach levels of mercury in blood close to or higher than 200 jug/L (EHC 101, 1990). Based on data from Iraq and data from fish consuming populations in New Zealand, a maternal level of 10-20 /ig Hg/g hair is estimated to impose a 5% risk of neurological disorder and to decrease test performance in the child (Kjellström et al., 1989; EHC 101,1990). A recent investigation on the Faroe Islands revealed that the mean value of Hg in maternal hair among mothers at 1024 consecutive deliveries was 6 /ig Hg/g (Grandjean et al., 1991). The data further indicate that almost 13% of the Faroe women giving birth have a mercury level in hair exceeding 10 /xg/g. Thus, in this population consuming large quantities of marine food, the body burden of mercury may reach a level that, according to present risk assessment documents (EHC 101,1990), is associated with increased risk of toxicity in a significant and sensitive part of the population. However, this risk assessment is to a large extent based on the Iraqi data, which were obtained after relatively short-term exposure to high concentrations. Moreover, the concomitant intake of selenium from marine food could change the retention as well as the toxicity of methylmercury. Risk assessment of mercury exposure in relation to speciation is until now unresolved. Toxicokinetics of Methylmercury in Experimental Animals

Most experimental studies on the toxicokinetics of methylmercury used rats. The species differences between humans and rats affecting the toxicokinetics of methylmercury are in a number of ways larger than the species differences between humans and mice. Thus, the ratio between levels of mercury in plasma and red blood cells is about 200 in rats, whereas it is only 20 and 10 in mice and humans, respectively (Vostal, 1972). This difference together with more general differences in the be-


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havior of methylmercury in erythrocytes (Naganuma et al., 1980) may to some extent explain the observation that the ratio between blood and brain levels of mercury is 100-200 times higher in rats than in humans. In contrast, the blood/brain ratio of mercury is only 5-10 times higher in mice than in humans (Berglund et al., 1971). Moreover, in contrast to other experimental animals and humans, the rat has no gallbladder, and other species differences in bile flow and biliary conjugates of methylmercury have also been reported (Klaassen and Watkins, 1984; Naganuma and Imura, 1984; Stein et al., 1988; Urano et al., 1988). These differences between rats and other experimental animals and humans may thus complicate comparisons of results obtained with rats with data obtained with other species. Orally administered methylmercury is almost completely absorbed in humans (Miettinen, 1973). Due to the lipophilicity of CH3HgCI, the intestinal absorption of methylmercury would be expected to be high, almost 100%, in experimental animals also, thus minimizing the differences between the toxicokinetics of orally and intraperitoneally administered CH3HgCI. Accordingly, earlier studies have demonstrated identical halftimes of methylmercury in mice exposed intraperitoneally and in mice fed a diet with CH3HgCI added (Clarkson, 1972). However, the available information on intestinal uptake of orally administered methylmercury in experimental animals is limited, and divergent figures for the wholebody retention of mercury at similar periods after dosage to mice have appeared (Clarkson, 1972; Landry et al., 1979; Mehra and Kanwar, 1980; Rowland et al., 1986). Furthermore, differences between retention of orally and intraperitoneally administered methylmercury have been reported (Mehra and Kanwar, 1980). The whole-body burden of a toxic metal is normally considered equal to the amount of metal available for inducing toxic effects. However, toxic metals may accumulate in inert compartments. Accordingly, part of the body burden may not be available for inducing toxicity. Thus, after exposure to methylmercury a considerable part of the body burden of methylmercury might with time be deposited in inert compartments such as hair. As mercury deposited in hair is without any toxic effect, it could be regarded as excreted. Differences in whole-body retention or elimination rate of mercury between animal species or strains might be caused either directly by different rates of excretion or indirectly by different rates of deposition in those compartments from which mercury is mobilized slowly or not at all. A perusal of the literature on mercury deposition in fat, muscles, skin, and fur after administration of CH3HgCI to experimental animals revealed that our present knowledge is limited. In relation to mice, published results are almost absent. Previously published investigations of the toxicokinetics of mercuric mercury (Hg +) and methylmercury (CH3Hg+) demonstrates several discrepancies, which might be due to incomparable experimental condi-


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tions in the different studies. Earlier studies did not systematically compare various experimental conditions influencing the toxicokinetics of methyl mercury. Therefore, the present study aimed at first establishing a rapid and reproducible experimental method for determining the disposition of various forms of mercury after different administration routes and second at using it for quantitative determination of effects of dose, route of administration, strain, and sex of mice on the toxicokinetics of mercuric mercury and methylmercuric chloride. The results add new knowledge allowing firm conclusions to be drawn concerning effects of strain, sex, dose, and administration route that were previously based on comparison of studies using different experimental methods.


METHODOLOGICAL CONSIDERATIONS

To investigate the toxicokinetics of mercury, a valid and accurate method is a prerequisite, and any shortcomings must be known in detail. Moreover, as the evaluation of toxicokinetics of mercury involves a large number of quantitative determinations in different organs, the resources must be used efficiently. Whole-body retention and organ deposition of a metal may be estimated from total body or organ loads or from concentrations within organs. The more classical procedures are performed with tissue samples and use elemental analysis or liquid scintillation counting a ßemitting isotopes. However, the use of 7-emitting isotopes in combination with a well-crystal enables consecutive countings of live animals with the purpose of describing the time course for elimination or deposition. Estimation of whole-body retention and intestinal absorption by whole-body counting has the advantage of employing the intact, unperturbed intestinal system with its natural contents and during normal function in the intact animal. However, this method does not measure flows but merely total deposition/retention. Thus, a rapid reexcretion of intestinally absorbed mercury would cause an underestimation of the intestinal absorption. This could be corrected in part by using data from parenterally administered mercury. The author previously used an experimental model in which a gamma-emitting isotope (109Cd) was administered to mice followed by consecutive whole-body countings, autopsy, and counting of organs (Andersen et al., 1988a, 1988b, 1988c, 1989a, 1989b; Andersen and Nielsen, 1988, 1989). This model was further refined to allow the study of the toxicokinetics of mercuric compounds. Animals All experiments used 7- to 8-wk-old mice kept on beechwood bedding in a well-controlled environment (50 ± 5% relative humidity, 20 air

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changes/h, temperature 21 ± 1°C, light/dark periods 12/12 h with 1A> hour twilight) with free access to standard mouse pellets (Brogaarden, Chr. Petersen, Ringsted, Denmark) and water. One strain of outbred mice (Bonr.NMRI) and three strains of inbred mice (C57BI/6JBom, C3H/TifBom, CBA/JBom) were used in the experiments.


Mercury Mercury was administered in the form of mercuric chloride (HgCl2) or methylmercuric chloride (CH3HgCI) (analytical grade, Merck, Darmstadt, Germany) labeled with 203HgCI2 or CH3203HgCI (Amersham, U.K.), respectively. As dissociation of soluble mercury compounds occurs, and as the mercury(ll) ion is entirely bound to proteins or other thiol containing ligands in vivo, the systemic transport and the excretion of absorbed mercury would be expected to be the same regardless of the anion in the soluble salt administered. Further, the rate of intestinal absorption of mercury from various soluble compounds would be expected to be similar, as these salts easily dissociate when dissolved in the gastrointestinal contents. 7 Counting The mercury isotope 203Hg is suited as tracer for mercury because it has a strong peak of y emission at 279 keV, which is within the optimum energy detection region of the Nal crystal in the gamma counter. The half-life of 46.6 d allows relatively long experimental periods, although the isotope decay causes decreased specific activity that must be taken into account throughout the experiment. This is normally accomplished by use of a 203Hg standard (volume 1 ml) with a known initial amount of Hg and a specific activity identical to that of the dosage solution. Relating all countings in an experiment to the standard will automatically adjust for isotope decay and enable accurate quantitation of mercury in whole body and organs. This is, however, based on the assumption that isotope dilution or isotope exchange does not occur to any significant extent. The assumption on which all tracer techniques are based is that the tracer and the tracée behave identically. Thus, the specific activity is assumed to be unchanged throughout the experimental period with the exception of natural decay of the isotope, which is corrected for as already described. To assure this basic requirement, the endogenous concentration of tracée must be close to zero, or at least insignificant, at the time of administration of the exogenous tracer and tracée. With most essential elements this is unachievable at physiologically relevant conditions. However, with mercury this assumption can be met by assuring that the preexperimental exposure to the metal is negligible. The amount of mercury in the commercial feed is, according to the manufacturer's description, below an analytical detection limit of 0.01 mg/kg feed (~50


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nmol/kg feed). This corresponds to a daily oral intake of 0.01 /imol mercury/kg (2 /ig Hg/kg) in a 25-g mouse eating 5 g/d. This maximum daily intake is below 1% of the lowest administered oral dose of HgCI2 and is therefore regarded as insignificant.


Validation of 7 Counting

As all data in this project are based on counts recorded by the 7 counter (Searle 1195R), it is essential to evaluate linearity of the countings, counting efficiency, effect of counting geometry, and self absorption, and to establish the limits within which counting results are valid. If ideal linearity existed, the counts should increase proportionally to the increased amount of isotope added to the vial. The ideal linearity was present up to approximately 3.5 x 106 cpm (Fig. 1). At higher amounts of isotope, the 7 counter was probably unable to register all the incoming signals and the linearity curve dropped below the ideal curve. Normally the mice were given an amount of Hg corresponding to initial counts between 5 x Iff and 1 x 106 cpm. The counting efficiency in both organ and whole-body counter was tested with a window that included the 279keV peak of the isotope. Calculated from the manufacturer's description of specific activity, this setting yielded a counting efficiency of 45% for a standard, which was considered satisfactory. As the different organs investigated from the mice vary in size between 0.2 and 2.5 g (intestinal tract often 4-5 g), it was essential to evaluate the effect of different sample volumes on the counting efficiency in the organ counter. Thus, increasing amounts of water were added to an initial amount of 203Hg in a vial. The counting geometry (Fig. 2) showed 10

I

200

400

600

800 1000 1200 1400 1600 1800 2000 amount of mercury (ul)

FIGURE 1. Linearity of the y counter and the two crystals (•, organ crystal and G, whole-body crystal) with respect to 203Hg was investigated by adding increasing amounts of the 7-emitting isotope to plastic vials.

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0,5

1.5

2 2,5 3 test volume (ml)

3,5

4,5

FIGURE 2. Counting geometry in the organ crystal with respect to 203Hg was investigated by increasing the test volume with water and keeping a fixed amount of 7-emitting isotope in the plastic vial.

that compared to a standard with a volume of 1 ml the maximum error due to sample size was 7.5% within sample volumes from almost zero to 2 ml (liver, stomach). The underestimation of the intestinal deposition could at maximum approach 25-30%. As the intestinal deposition was only compared between different groups in which the same degree of error due to underestimation could be anticipated, no correction in counting data was made. The relatively low energy (279 keV) will result in volume-dependent counting due to different absorption in air and tissue/water. In order to evaluate the absorption in the sample counted in the whole-body counter, fixed amounts of 203Hg were dispensed into small plastic vials and counted. Then the plastic vials were placed inside a dead mouse and recounted. The absorption in mouse tissues was estimated to be about 4.5% CTable 1). Whole-body counts were not corrected with this factor. TABLE 1. Estimation of Self-Absorption of 203Hg in Whole-Body Counter Using Phantoms

Test vial

7 Counting of vial (cpm)

7 Counting of vial inside mouse (cpm)

1 2 3 4

131,650 132,149 66,800 67,050

125,470 124,668 64,008 64,149

Self-absorption (%)

4.69 5.66 4.18 4.33 Median value 4.5

Note. Two different amounts of mercury were used but given in identical volumes (0.2 ml).


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To define an interval in which counting results are valid requires setting of two limits. A maximum amount of 7 emission that can be counted accurately has already been mentioned. The lower limit is determined by the background and its variation. The backgrounds in the whole-body and organ counters were about 400 and 50 cpm, respectively. A lower limit for using counting results was set at the average background level plus three standard deviations (based on 60 background countings). The standard deviations of the background countings were normally in the range of 8-15 cpm, causing a lower limit for accepting results of about 445 cpm in the whole-body counter and about 74 cpm in the organ counter. The detection limit expressed in absolute terms (pmol Hg) will of course vary from experiment to experiment depending on the specific activity of the purchased batch of Hg. However, the lower level for detection was between 0.4 and 2.4 pmol mercury in experiments with HgCI2 and between 40 and 240 pmol mercury in experiments with CH3HgCI. Whole-Body Retention

For each animal, the whole-body retention of the dose was expressed as percent of its initial count. The time needed for elimination of 70% of the oral dose (T07) quantifies delayed initial elimination and is used as a measure of toxic effects on the peristaltic process. Earlier studies of the toxicokinetics of orally administered cadmium have demonstrated that the time for elimination of the major part of a nonabsorbed toxic dose of a metal is a quantitative measure of the toxicity toward the peristaltic process (Andersen et al., 1988a). The T07 was estimated from the wholebody counting curves. Organ Distribution

At the end of an experimental period animals are killed, dissected, and relevant organs and tissues are removed, weighed, and counted. The organs normally used are liver, kidneys, stomach, intestinal tract, testes or ovaries with uterine horns, heart, spleen, lungs, brain, and the remaining carcass. However, in relation to methyl mercury, which to a large extent is deposited in carcass, the content of mercury in blood, hair, fat, bone, and skin was also measured (described in Nielsen and Andersen, 1991b). Each animal's organ counts were expressed as percent of the wholebody retention at the day the animals were killed. The formula given next eliminates any correction for possible differences in counting efficiencies in whole-body and organ counter, and use the countings (cpm) of organs, whole body, and carcass directly after correction for background.

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. .. . .i. organ(n) x (100 - carcass/whole body) 0/ Relative organ deposition = —Q *- % 5 K /0 organd) + • • • + organ(9) This proportion is a measure of the fraction of the residual body burden present in the organ, that is, the relative distribution of mercury in different compartments. The use of relative values for evaluating the organ distribution of retained mercury has certain advantages. First, as the dose (/*mol/kg body weight) is determined by the size of the animals, variations due to differences in size of animals are eliminated. Second, as the amount of mercury in various organs would increase with increasing dose, statistically significant differences in organ concentration and absolute organ deposition would inevitably occur in experiments with different doses. However, these differences would not necessarily be related to differences in fractional organ deposition but rather to the magnitude of the dose. In experiments with different doses, dose-related differences in relative organ distribution are easily recognized when the distribution is expressed in relative terms. Even in oral exposure experiments where identical doses are given, variations in intestinal uptake due to interexperimental variation or different treatment (i.e., diet, chelating agents) would cause different organ loads of Hg, which could be more related to internal dose size than to disposition. Total organ depositions are easily calculated by relating the actual organ count (corrected for background) to the standard of known intensity (cpm/pmol Hg). The organ concentration can be calculated by dividing by the organ weight. Absolute values are preferentially used in cases where the total organ, tissue, or body fluid is practically unavailable for analysis (blood, fat, bone, etc.). Moreover, absolute values are of interest in relation to toxicity studies in which thresholds for adverse effects can be calculated and in experiments where animals are treated identically except for a moderator of toxicity (i.e., chelating agents, selenium vs. mercury toxicity).


n

Perfusion of Animals

The use of organs from unperfused animals will cause the blood present in each organ to be counted together with the organ in the organ counter. Previous use of the experimental model to study cadmium toxicokinetics did not require any precautions with respect to cadmium in the blood, because of only limited amounts of cadmium in blood. However, mercury and especially methylmercury are present in blood for substantial time periods after exposure. To investigate the effect of mercury in blood, groups of mice were given a single oral dose (5 /zmol/kg) of either HgCI2 or CH3HgCI and killed after different periods (1, 2, 3, 10, 20, 30 d). Blood was removed from the thoracic cavity (~1 ml/mice). Mercury deposition in blood after HgCI2 administration de-



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dined rapidly, and at d 30 the amount was below the detection level of the organ counter (Fig. 3). The normal experimental period is 14 d and at this time approximately 2% of the initial blood level was still present in the mice given inorganic mercury. The level of mercury in the blood after CH3HgCI administration declined much more slowly, and at d 14 the blood level of mercury was still around 25% of the value at d 1 (Fig. 3). Therefore, it was necessary to evaluate the importance of perfusion on results from organ countings. Two groups of male mice (Bom:NMRI, oral administration) and two groups of female mice (BomrNMRI, intraperitoneal administration) were given the same dose (5 /*mol/kg) of CH3HgCI. At d 14 one group of each sex was immediately killed, whereas the other two groups were initially anaesthetized with Nembutal and perfused with Ringer's solution to remove blood from the organs before y counting. Data from the two groups perfused were almost identical to those of similarly treated nonperfused groups, although a tendency to higher relative deposition of mercury in brain and utérines of females and a reduced relative deposition of mercury in male kidneys and heart were observed (Nielsen and Andersen, 1991a). Thus, the effect of perfusion before removal of organs for 7 counting was insignificant in the present experiment terminated at d 14. However, in single-dose experiments terminated early after dosage or in chronic exposure experiments, the mercury in the blood may influence determination of the relative organ distribution. Thus, if the blood contains significant amounts of mercury, the relative deposition of mercury in organs with high amounts of blood will be overestimated in animals that are not perfused, whereas the relative deposition in organs

100004:^:::::::::

FIGURE 3. Amount of mercury in blood during a 30-d experimental period after a single oral dose of HgCI2 (D) or CH3HgCI (•).

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with relatively low contents of blood will be underestimated. In the present experiment with perfused animals, the slightly higher relative deposition in brain and uteri of female mice was in agreement with the normally relative low content of blood in these organs, and the opposite effect was seen in male kidney and heart.


Reproducibility Comparison of data within and between experiments requires knowledge about the interexperimental as well as the intraexperimental variation. The intraexperimental variation was investigated as part of a timecourse analysis for organ distribution in which 12 groups of female mice were initially given the same oral dose of mercury (HgCI2 or CH3HgCI). Whole-body counting was repeated at d 1, 2, 3,10, 20, and 30 (one group of mice given HgCI2 and CH3HgCI was killed for organ analysis on each of these days). These data on whole-body retention demonstrate excellent intraexperimental reproducibility of the whole-body counting method after oral administration of CH3HgCI or HgCI2 (Figs. 4 and 5). Similar agreement between whole-body data within an experiment using the present experimental model was previously demonstrated after oral cadmium administration (Andersen et al., 1989a) and intraperitoneal CH3HgCI administration (Nielsen and Andersen, 1991a). Previous experiments with cadmium have demonstrated that day-today variations do occur even in experiments with the same strain of inbred mice maintained under identical conditions. Thus, in independent experiments with identical doses, whole-body retention of orally administered cadmium at d 10 after dosage varied between 0.74 and 1.83% (Andersen et al., 1988a, 1989a). The same degree of variation was Intra-experimental variation methylmercury (5 u.mol/kg) WBR (%) 100i

Days FIGURE 4. Intraexperimental variation of whole-body retention after methylmercury administration was assessed after administering CH3HgCI to 6 groups of 10 mice. Whole-body retention was followed until the group was sacrificed for organ analysis.


99

Intra-experimental variation mercuric mercury (5 u.mol/kg)

WBR (%) 100-3

0,1 Days

10

20


FIGURE 5. Mercuric mercury (HgCI2) intraexperimental variation as in Fig. 4.

observed in experiments with HgCI2. Thus, in 6 experiments with identical HgCl2 doses (5 /xmol/kg) over a period of 2 yr, whole-body retention at d 14 was found to be 0.92,1.11,1.20,1.27,1.35, and 2.05% with a median value of 1.24%. Therefore, comparisons between different experiments must take into account the possible interexperimental variation.

Statistics The group sizes in the present experiments were either 10 or 20 mice. In the latter situation, the counting values from the different animals in each experimental group fitted an approximate gaussian distribution and it was considered appropriate to compare group means by Student's ttest (two-sided). In the case of 10 animals per group, an approximated normal distribution could not be certain and results were compared using the nonparametric Mann-Whitney rank test. In both cases, the level for rejection of the Ho hypothesis was set at .05. RESULTS AND DISCUSSION

Previous investigations studying quantitatively the toxicokinetics of inorganic and organic mercury are relatively few, and most employed parenteral administration of a soluble mercury salt. The limited information available is briefly reviewed below and discussed in relation to main results from the present investigation (Nielsen and Andersen, 1989,1990, 1991a, 1991b). Whole-Body Retention and Elimination

Mercuric Chloride Mice given 100 /tmol/kg of HgCI2 orally had a significantly decreased elimination of mercury within the first 3-4 d after


100

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dosing compared to mice given 1, 5, or 25 /imol/kg (Figs. 6 and 7). The reduced elimination rate due to peristaltic delay was visualized by the "shoulder" (delay) on the whole-body counting curve for both male and females (Figs. 6 and 7) and by an increased 707 in the groups given 100 /^mol/kg HgCI2 orally compared to the groups given the lower doses (Table 2). A similar effect on peristalsis was previously observed after oral exposure to CdCI2 (Andersen et al., 1988a). In the cadmium study, postmortem examination showed intestinal atony and histological investigation demonstrated severe tissue damage to the gastrointestinal tract. Although the doses of HgCI2 employed in the present study induce less toxicity than the doses of CdCI2 used in our previous studies, the larger T07 values measured on whole-body counting curves of the groups given the highest dose of HgCI2 (Table 2) indicated an increased fecal passage time due to peristaltic toxicity of HgCI2. The initial fecal elimination rates at the lower doses (T07 = 17-21 h) corresponded to those found in our previous studies after administration of the lowest doses of CdCI2 (Andersen et al., 1988a). Comparison of results from female mice with those from male mice demonstrates that the sex of the mice affects the whole-body retention of orally administered HgCI2. The female mice (both.Bom:NMRI and CBA/JBom, Table 2) seem to retain considerably less mercury than do male mice. A similar sex difference in whole-body retention of methylmercury was previously observed in adult mice and rats (Inouye et al., 1986; Thomas et al., 1987). Although the mechanism is unknown, testosterone levels were suggested as a possible modifying factor as the difference was observed in adult animals only (Hirayama and Yasutake, 1985). Compared to initial excretion rates measured in experiments using parenteral administration of inorganic mercury, the initial elimination rates measured in this study using oral administration are of course higher, as they pertain mainly to fecal elimination of nonabsorbed mercury. This does not occur after intraperitoneal administration of HgCI2, but nevertheless, the initial elimination rate after parenteral administration was larger than that during the later part of the experimental period (Fig. 8). This initial fast elimination phase, which was also demonstrated in earlier investigations (Rothstein and Hayes, 1960; Kristensen and Hansen, 1980; Daston et al., 1986), could be due to an initial rapid excretion of absorbed mercury through liver and bile into feces and through kidneys into urine before deposition of Hg at firmer binding sites. Extrapolating back to zero time from the experimental period after initial fecal elimination of nonabsorbed mercury had taken place indicated initial excretion rates in the same order of magnitude as those measured after parenteral exposure. In contrast to observations after oral administration of CdCI2 indicating increased fractional retention with increasing dose (Andersen et al., 1988a), the fractional whole-body retention of HgCI2 at d 14 after dosage


101

100 -i

QJ


O

0.110 Days FIGURE 6. Effect of dose of HgCI2 on whole-body retention of mercury after a single oral dose of HgCI2 to male Bom:NMRI mice. The standard error of the mean is less than the height of the symbols: (A) 1 nmol/kg, n = 20; (O) 5 jtmol/kg, n = 19; (V) 25 /*mol/kg, n - 19; (D) 100 /^mol/kg, n - 19. From Nielsen and Andersen (1989).

is inversely related to the dose. The reason for this dose dependency might be saturation of the intestinal uptake mechanism at high dose levels (100 /*mol/kg). Another explanation could be damage to the kidneys resulting in the loss of mercury with the urine, which is the main excretion route after exposure to high levels of inorganic mercury (Kristensen and Hansen, 1980). However, this would require that the excretion rate indicated by the later part of the elimination curves should demonstrate a positive dose-response relationship. This is suggested in the present study by the half-times seeming to decrease with increasing dose. However, the present study demonstrates that this inverse relationship cannot be due to saturation of the intestinal uptake mechanism at high doses, as the same relationship was also observed after intraperitoneal administration of HgCI2. Damage to the kidneys due to high levels of inorganic mercury has previously been described after intravenous administration of high doses of inorganic mercury (2-6 ^mol Hg/kg) to rats (Piotrowski et al., 1969). Furthermore, rather low doses of mercuric

). B. NIELSEN

102

100-

10-


1-

0.17 Days

I

10

14

FIGURE 7. Effect of dose of HgCI2 on whole-body retention of mercury after a single oral dose of HgCI2 to female CBA/JBom mice: (A) 1 /*mol/kg, n - 10; (O) 5 /^mol/kg, n - 10; (V) 25 /^mol/kg, n = 10; (D) 100 /tmol/kg, n = 10. From Nielsen and Andersen (1990). TABLE 2. Effect of Dose on Whole-Body Retention of Mercury at Day 14 After Administration of 203 HgCI2 (% of Administered Dose) and T07 (h) as Calculated from Whole-Body Counting Curves CBA/JBom

Bom :NMRI Male

Female

Male

Female

Dose 0imol/kg)

TOj

WBR

To.7

WBR

T0.7

WBR

7-0.7

WBR

1 5 25 100

19 18 21 37

2.8 1.9 1.7 1.2

18 18 19 41

1.3 0.8 0.7 0.8

17 17 19 31

1.7 1.5 1.6 1.0

19 19 18 36

1.1 1.0 0.6 0.7

Note. T07 is the time (h) needed for elimination of 70% of the initial dose of 203HgCI2 (Nielsen and Andersen, 1989, 1990). Results are given as medians of 10 mice per group. WBR, whole-body retention.


103

100-

10o T3


1-

0.1 7 Days

10

FIGURE 8. Effect of dose of HgCI2 on whole-body retention of mercury after a single intraperitoneal dose of HgCI2 to female CBA/JBom mice: (A) 0.05 jimol/kg, n = 9; (O) 0.25 /xmol/kg, n - 10; (V) 1.25 /^mol/kg, n = 10; (D) 5.00 fjmol/kg, n - 10. From Nielsen and Andersen (1990).

salts are able to affect the kidney function as reflected by an increased diuresis and decreased reabsorption of amino acids (Clarkson, 1968). Accordingly, renal tubular damage and increased urinary excretion of mercury after oral administration of 100 /xmol/kg HgCI2 to female BonrNMRI mice have been demonstrated (Nielsen et al., 1991). The decreased initial elimination rate seen after the highest oral dose of HgCI2 was not observed after the highest intraperitoneal dose (Fig. 8). In contrast, the initial elimination rate of intraperitoneally administered HgCI2 was highest at the highest dose. The rate of excretion of systemic mercury as estimated from the last part of the whole-body counting curves was found to be independent of the dose within the dose range used in these experiments, except that Bom:NMRI mice eliminated the absorbed part of the highest oral dose more rapidly (Nielsen and Andersen, 1990). The excretion rate was similar in the two mice strains after oral or intraperitoneal administration of HgCI2. Both mouse strains excreted intraperitoneally administered HgCI2 slower than orally administered HgCI2. The larger elimination rate during the last week of the ex-


104

). B. NIELSEN

perimental period in groups given HgCI2 orally compared to groups given HgCI2 intraperitoneally cannot be due to differences in the size of the systemically delivered dose of mercury, as the apparent half-time seems to be independent of the dose size within the dose range used in this study. Furthermore, calculations of the absolute amounts of mercury retained at 14 d after dosage show that groups given 25, 5, or 1 /*mol/kg orally retain amounts of mercury comparable to that in groups given 5, 1.25, 0.25 ^imol/kg intraperitoneally, respectively. Therefore, the difference in the excretion rate between orally and intraperitoneally administered mercury is more likely due to differences in the disposition of systemically delivered and retained mercury between these two administration routes. This was confirmed by studies of the organ distribution of retained mercury (see section on organ distribution below). The fractional whole-body retention is not at any time after oral dosage an accurate measure of the fractional intestinal absorption of mercury because part of the absorbed mercury will be rapidly excreted during the experimental period. The fractional whole-body retention of intraperitoneally administered mercury can be used to estimate the fractional retention of orally absorbed mercury and thus allow calculation of the "true absorption" (At) (Van Barneveld and van den Hamer, 1986): whole-body retention (oral) whole-body retention (ip) Although this procedure may introduce a bias because differences do exist in the toxicokinetics between orally and intraperitoneally administered mercury, this correction will give a more accurate estimate of the intestinal uptake of mercury. Using whole-body retention data after oral and intraperitoneal administration, the estimated "true absorption" of a single oral dose of HgCI2 (1 and 5 /nmol/kg) may be calculated to be 20-25% in mice. As the apparent half-time of retained mercury is shorter in orally exposed mice than in intraperitoneally exposed mice, the bias introduced will tend to decrease the estimated gastrointestinal absorption. The absorption of orally administered HgCI2 has previously in a number of studies been estimated to vary between 7 and 15% (Ellenhorn and Barceleux, 1987). The reexcretion of absorbed mercury during the experimental period was not taken into account in these estimates that were based on the total amounts of excreted mercury. The large difference in intestinal uptake of mercury calculated from data of this study compared to previous studies has wide implications for risk assessment in relation to oral exposure to inorganic mercury. The conclusions of the present study therefore should inspire similar studies in other species.

105



Methylmercuric Chloride Generally, the whole-body retention curves after CH3HgCI exposure (Nielsen and Andersen, 1990a) (data only shown for female Bom:NMRI mice, Fig. 9) are almost straight lines in a semilogarithmic diagram, indicating apparent first-order elimination kinetics for both orally and intraperitoneally administered CH3HgCI during a 2-wk study period. Comparison of whole-body retentions in female mice given CH3HgCI either orally or intraperitoneally demonstrated, in agreement with results published by Clarkson (1972), no significant differences in whole-body retention at d 14 and similar half-times calculated from the whole-body counting curves (Table 3). The data on whole-body retention from orally and intraperitoneally exposed mice may be used for calculation of the "true" absorption, Au of orally administered CH3HgCI. In the present study, Ax of orally administered CH3HgCI in female mice at all 3 doses was calculated to vary between 97 and 101%, which is in agreement with the literature describing almost complete absorption. The effect of dose on whole-body retention at d 14 and tv (Table 3)

5! o

10-

1

I

I

I

2

3

4

7 Days

1

10

FIGURE 9. Effect of dose of CH3HgCI on whole-body retention of mercury after a single oral dose of CH3HgCI to female Bom:NMRI mice: (A) 0.2 jjmol/kg, n - 10; (O) 1.0 /imol/kg, n - 10; (V) 5.0 /imol/kg, n - 10; (D) 25.0 ^mol/kg, n - 10. From Nielsen and Andersen (1991).

106

). B. NIELSEN


TABLE 3. Effect of Dose, Sex, Route of Administration, and Strain on Whole-Body Retention (WBR%) at Day 14 and Tentative Half-Time (d) Calculated from the Whole-Body Retention Curves After a Single Dose of Methylmercuric Chloride to Mice WBR at d 14

Elimination f,

Sex

Route of administration

Dose

Strain

(/^mol/kg)

(%)

(d)

Bom:NMRI Bom:NMRI Bom:NMRI Bom:NMRI Bom.-NMRI Bom:NMRI Bom:NMRI Bom:NMRI Bom:NMRI Bom:NMR! CBA/JBom C57Bl/6JBom C3HffifBom

F F F F F F F M M M M M M

Intraperitoneal Intraperitoneal Intraperitoneal Oral Oral Oral Oral Oral Oral Oral Oral Oral Oral

1.0 5.0 25.0 0.2 1.0 5.0 25.0 1.0 5.0 25.0 5.0 5.0 5.0

47 (43-51) 44 (41-47) 37 (34-39) 53 (41-63) 44 (37-49) 44 (37-49) 36 (29-39) 23 (18-31) 26 (18-35) 15 (10-19) 37 (35-40) 21 (19-28) 18 (18-20)

13 13 10 16 13 13 10 9 9 7 9 6 6

Note. Group size — 10. Results for WBR% are given as medians with 25th and 75th percentiles (Nielsen and Andersen, 1991a, 1991b).

indicates an inverse relationship between dose and whole-body retention and between dose and U^. A similar relationship was demonstrated in mice orally exposed to HgCI2 and was not due to an effect on the intestinal uptake mechanism (discussed earlier). Nor in the experiments with CH3Hg+ was the inverse relationship between dose and whole-body retention caused by an effect on the uptake mechanism as complete intestinal absorption was demonstrated at all doses. The half-times in both males and females decrease with increasing doses within the dose range used. This is in agreement with and further extends an earlier study indirectly suggesting this dose dependence by demonstrating an inverse relationship between dose size and half-time of methylmercury in various organs (Suzuki, 1969). Sexual differences in whole-body clearance and tissue distribution of intestinally absorbed methylmercury have been demonstrated in rats, mice, and humans (Magos et al., 1981; Miettinen, 1973; Thomas et al., 1986; Yasutake and Hirayama, 1988). Furthermore, differences in wholebody retention between male and female mice depend on the mouse strain. Thus, male C57BL/6N mice excreted intestinally absorbed methylmercury much faster than female mice of the same strain and survived longer after a lethal oral dose of methylmercury. In contrast, males of the BALB/cA strain were more susceptible to the toxicity of oral methylmercury than the females of the same strain (Yasutake and Hirayama, 1988). In the present study, male mice excreted methylmercury significantly faster than females, and at d 14 the whole-body retention of mercury in



107

the males was only about 50% of the whole-body retention of mercury in females. This is also illustrated by a significantly higher f1; j n female mice (10-13 d) compared to males (7-9 d). The t^ values found in this study are generally larger than those previously reported. Thus, different studies found ty^ values for methylmercury in mice between 6 and 9 d (Clarkson, 1972; Greenwood et al., 1978; Landry et al., 1979). Toxicological studies are often performed using only a single inbred strain of an experimental animal, thus assuming only limited or even absence of differences in toxicokinetics between strains (Festing, 1990). Previous studies on toxicity and toxicokinetics of methylmercury in mice described strain-related differences in rate of urinary excretion and amounts of mercury deposited in blood, liver, kidney, and brain (Doi et al., 1983; Yasutake and Hirayama, 1986,1988). The present study extends these findings by demonstrating differences in both whole-body retention and rate of whole-body elimination of mercury after oral administration of methylmercury to three strains of inbred mice. The present study demonstrates that the dose, sex, and strain significantly affect the whole-body retention and half-time of mercury after administration of CH3HgCI. Finally, the route of administration, whether oral or intraperitoneal, does not to any appreciable degree affect the whole-body retention of methylmercury within the dose ranges used in these experiments. Organ Distribution

Mercuric Chloride The relative organ distribution in selected organs after exposure to HgCI2 by the oral or the intraperitoneal route is shown in Figure 10. Retained Hg was mainly deposited in kidneys, liver, and carcass. While the relative organ distribution (i.e., the fraction of absorbed and retained mercury in a specific organ at the time of analysis) did not vary systematically with dose in carcass, lungs, and brain, increased relative deposition with increasing dose was observed in all other organs studied, except in the kidneys (Nielsen and Andersen, 1990a). The relative deposition in liver increased with the dose size in both BOITKNMRI and CBA/Bom mice (Fig. 10). The relative deposition in the liver was doubled from the lowest to the highest dose in orally exposed female Bom:NMRI mice, whereas the increase was more limited in male mice. Within the dose range used, the relative deposition in kidneys after oral exposure decreased considerably more in female Bom:NMRI mice than in CBA/JBom mice. Irrespective of dose and route of administration, the deposition in carcass accounts for approximately 25-35% of the residual body burden at 2 wk after a single dose in female and male mice (Fig. 10). The ratio between the relative deposition of orally administered

J. B. NIELSEN

108

6030CO

I

Q.

40-H

20-

—

10-

-

I 20 •33

— 0-

ti,2.5 q

n

Q.


O

bra

i

r-i

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|

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'55 0 Q.

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0CBA (m,o)

NMRI (m,o)

NMRI (f.o)

NMRI

.2 30 .*; (0 o

| 20-H

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1 |imol/kg (oral), 0.05 nmol/kg (ip)

[]]

25 nmol/kg (oral), 1.25 nmol/kg (ip)

[23

100 nmol/kg (oral), 5 |imol/kg (ip)

rr CBA ' NMRI ' NMRI ' NMRI (m.o) (m,o) (f.o) (f.ip) FIGURE 10. Effect of dose, sex (m, male; f, female), strain (CBA/JBom or Bom:NMRI), and route of administration (o, oral; ip, intraperitoneal) on relative organ distribution of mercury (percent of whole-body retention at d 14) after a single dose of HgCI2. Results are given as medians (group size - 10) (Nielsen and Andersen, 1989, 1990).

HgCI2 in liver and kidneys of female Bom:NMRI mice changed from 0.4 to 1.3 when the dose was increased from 1 to 100 /*mol/kg, but the combined relative deposition in liver and kidneys was constant between 56 and 58% at all dose levels (Fig. 10). A similar combined deposition in liver and kidneys (54-60%) was found in female CBA/JBom mice after oral administration of HgCI2, but the liver/kidneys ratio was not changed to



109

the same extent (data not shown), although the relative deposition in the liver reached the same level as in the kidneys. The relative deposition of intraperitoneally administered HgCI2 in liver increased somewhat with the dose size, but the increase in the relative deposition in the reproductive organs was considerably higher (Fig. 10). The relative deposition of intraperitoneally administered HgCI2 in kidneys and brain decreased with increasing dose, while the relative deposition in carcass remained constant within the dose range used. The increase in relative deposition in the liver and the decrease in relative kidney deposition at increasing intraperitoneal HgCI2 doses led to an increase in the ratio between the mercury burden in liver and kidneys from 0.2 to 0.6. However, except for the carcass, the kidneys remained the major depot for mercury. Previous studies have demonstrated that at 2 wk after dosage the major part of the mercury was present in the kidneys after oral as well as parenteral exposure (Berlin et al., 1966; Rothstein and Hayes, 1960). Thus, 2 wk after a single intravenous injection of 0.05 /^mol/kg b.w. Hg(NO3)2 to mice the mercury content in kidneys, liver, brain, and testicles was 19.3, 1.3, 0.7, and 0.8% of the residual body burden (Berlin et al., 1966). The relative kidney and liver deposition in mice given comparable nontoxic doses of inorganic mercury is reported to be approximately 2 and 10-15 times higher, respectively, after oral administration than after intravenous injection (Berlin et al., 1966). In contrast, approximately 85% of the residual body burden was found in the kidneys and only 15% in the rest of the body 2 wk after a single intravenous injection of Hg(NO3)2 to rats (Rothstein and Hayes, 1960). How these obviously conflicting results could be explained is unclear, except for the possible effect of different animal species in these experiments. However, the differences in toxicokinetics of mercury between mice and rats described in several experimental reports (Berglund et al., 1971; Naganuma et al., 1980; Vostal, 1972) would not be expected to cause such substantial differences. In the only study using long-term oral exposure, the amount of mercury in rat kidneys after exposure to HgCI2 in the drinking water for 37 d was approximately 35 times larger than in the liver, corresponding to a 100-fold difference in concentration on the basis of organ wet weight (Mengel and Karlog, 1980). Comparison of the available data leads to the conclusion that the organ distribution of mercury after oral or parenteral administration of inorganic mercury is different both in terms of relative organ distribution and in quantitative terms due to different whole-body retentions. The differences in the toxicokinetics of orally and parenterally administered inorganic mercury are mainly due to the first-pass effect. Thus, the largest consistent difference in relative organ distribution after the two routes of exposure occurred in the relative liver deposition and in the ratio between


110

J.B.NIELSEN

hepatic and renal deposition of mercury. These differences were most clearly seen in the experiments with Bom:NMRI mice. The lower relative deposition in uteri observed in groups of female NMRI mice dosed orally compared to mice given HgCI2 intraperitoneally was not found in the CBA strain, in which the oral administration of HgCI2 as compared to intraperitoneal administration, on the contrary, significantly increased the relative deposition in utérines (Nielsen and Andersen, 1990). The relative testicular deposition was almost constant at the lower doses, but increased considerably at the two highest doses (Fig. 10, data from 5 /imol Hg/kg are not shown). The extensive increase in relative testicular deposition of mercury when the dose was increased from 5 to 25 fimoUkg indicates a susceptibility of the testes to orally administered HgCI2 at doses higher than 25 /*mol/kg. Inhibition of spermatogenesis was previously observed after intraperitoneal injection of 1 mg/kg HgCI2 to mice (Lee and Dixon, 1975), and the interstitial cells of the testicles accumulate mercury (Berlin and Ullberg, 1963). The relative deposition of orally administered HgCI2 in the brain did not depend on the dose size (Fig. 10). The brain would not be expected to accumulate significant amounts of inorganic mercury because of the blood-brain barrier. The present study demonstrates that between 0.51 and 0.92% of the residual body burden of mercury in males and up to 1.6% in females is retained in the brain at 14 d after dosage. Comparing the relative deposition of mercury in the brain to the relative deposition in other organs (i.e., stomach and lungs) and considering the different organ weights, the concentration of mercury in the brain at the end of the experimental period was at an unexpectedly high level. A minor deposition of mercury in the brain was also observed after a single intravenous injection of mercury into mice (Berling and Ullberg, 1963). As an accumulation of mercury in the brain across the blood-brain barrier during the 2-wk period after dosage seems unlikely, the higher concentration could more likely be due to either a slower excretion of mercury initially deposited in the brain than in other organs or a selective deposition of inorganic mercury in the brain during the initial distribution phase. Time-course analysis of the deposition of mercury in the brain during the initial distribution phase after a single oral dose of HgCI2 demonstrated an increasing relative deposition during the first 3 wk of observation. However, calculated as absolute amount of Hg, the deposition of mercury in the brain decreased very slowly from a maximum reached already at d 1 after exposure (Nielsen and Andersen, 1991c). The relative organ distribution did not show any major differences between mice of the Bom:NMRI and CBA/JBom strains, but mice of the CBA/JBom strain retained relatively more mercury in kidneys, intestines, and brain at all four doses than did mice of the Bom:NMRI strain. The residual body burdens and the relative organ distributions of mercury in



111

the two mice strains Bom:NMRI and CBA/JBom indicate no large differences, although statistically significant differences were observed in a few organs at some of the dose levels. The relative organ distribution in CBA/JBom mice did not change to any significant extent with increasing intraperitoneal doses of HgCI2 as observed in Bom:NMRI mice. The relative deposition in liver, kidneys, and the ratio between the deposition in liver and kidneys did not depend on the dose level. The quantitative changes were small in the CBA/JBom strain compared to Bonr.NMRI mice. Generally, dose-related changes in the relative organ distribution were more pronounced in the Bom:NMRI strain than in the CBA/JBom strain within the dose range used in these experiments. The distribution of mercury within the body and within specific organs varies with dose and time after exposure (Berlin, 1986). Furthermore, the route of administration affects the organ distribution of absorbed mercury (Miyama et al., 1968). The kidney has been described as the main depot after intravenous injection of HgCI2 (Gabard, 1976; Hayes and Rothstein, 1962; Rothstein and Hayes, 1960). At 2 d after subcutaneous administration of inorganic mercury, the kidney concentration was about 10 times that in liver and blood (Kristensen and Hansen, 1980). On the basis of earlier investigations, the kidney is considered the most important depot for mercury irrespectively of the administration route, with the liver as the second largest depot (Berlin, 1986). The present study demonstrates that this conclusion pertains to parenteral exposures only. Thus, the present study demonstrates that the toxicokinetics of orally administered inorganic mercury is different from the toxicokinetics of parenterally administered inorganic mercury as reported in the literature. As human mercury exposure is often via the oral route, a toxicokinetic analysis emerging from an experimental model using this exposure route is certainly of relevance for risk evaluation in relation to human mercury exposure. Methylmercuric Chloride The effect of dose on the relative organ distribution in female mice given CH3HgCI orally was minimal, although a tendency for a decreased relative deposition in the liver at increasing dose was observed (Fig. 11). The tendency was stronger and statistically significant in the groups given CH3HgCI intraperitoneally. An effect of dose on relative organ distribution was not observed in male mice (Fig. 11). The relative organ distribution of mercury varied significantly between the different strains of mice (Nielsen and Andersen, 1991). The significantly higher relative liver and kidney deposition of mercury in CBA/JBom mice than in C57Bl/6JBom (Table 4) concomitant with the significantly higher whole-body retention at d 14 (Table 3) causes a more than threefold higher absolute amount of mercury in liver and kidneys in CBA/JBom mice than in C57BI/6JBom mice. Also, the absolute deposition of mercury in the brain in mice of the CBA strain is twice the brain

J. B. NIELSEN

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Female.or Female.ip 1u.mol/kg

E3 5umol/kg

Male.or

Female.or Femalejp

H3 25(imol/kg

FIGURE 11. Effect of dose, sex, and route of administration (or, oral; ip, intraperitoneal) on relative organ distribution of mercury (percent of whole-body retention at d 14) after a single dose of CH3HgCI to Bom:NMRI mice. Results are given as medians (group size - 10) (Nielsen and Andersen, 1991a). TABLE 4. Relative Organ Distribution of Mercury (Percent of Whole-Body Retention at Day 14) After a Single Oral Dose of Methylmercury Chloride (5 jimol/kg) to 3 Strains of Inbred Mice and 1 Outbred Strain Organ

CBA/jBom

C57BI/6JBom

C3H/TifBom

Bom:NMRI

Liver Kidney Intestines Brain Carcass

7.54 13.65 3.66 0.76 72.50

3.62 (2.08-5.76) 6.96 (4.20-11.09) 2.32 (1.33-4.45) 0.72 (0.45-1.40) 85.06 '(73.97-90.98)

6.44 (4.86-7.67) 9.62 (7.05-11.14) 2.49 (2.02-2.89) 0.73 (0.55-0.78) 79.34 (76.38-84.60)

7.45 20.15 3.81 1.03 64.80

(6.86-8.35) (12.29-16.24) (3.23-4.07) (0.70-0.89) (68.62-75.22)

(6.04-8.17) (15.84-21.87) (3.10-4.23) (0.81-1.28) (61.78-71.94)

JVofe. Results are given as medians of 10 mice per group with 25th and 75th percentiles (data from Nielsen and Andersen, 1991a).



113

deposition in mice of the C57BI or C3H strain. Further, mice of the CBA strain have a significantly lower relative carcass deposition than the C57BI and the C3H strain (Table 4). Such differences may have implications for extrapolation based on studies using only a single inbred strain (Festing, 1990). Comparison of the toxicokinetics of methylmercury between the three inbred strains of mice and the outbred Bom:NMRI mice (males, 5 jimol/kg) demonstrates that although the whole-body retention at d 14 is not significantly different between Bom:NMRI and the two inbred strains C57BI/6JBom and C3H/TifBom, the relative organ distribution differs significantly (Table 4). Thus, the relative kidney deposition is twofold higher in the outbred than in these two inbred strains of mice. Previous studies on the effect of sex on organ distribution of mercury after methylmercury administration agree on a reduced deposition of mercury in male mice in all organs except in the kidneys when compared to the deposition in females (Hirayama and Yasutake, 1986; Yasutake and Hirayama, 1988). In the study of Yasutake and Hirayama (1988), a reduced deposition in the kidneys of males was observed in the C57BL/6N strain when compared with females of the same strain, whereas a slightly increased deposition in the kidneys of males was observed in the BALB/cA strain when compared with females of the same strain. In Bom:NMRI mice there was an increased relative deposition in female carcass, liver, stomach, intestines, heart, spleen, lungs, and brain and a considerable decrease in relative deposition in female kidneys (Fig. 11) (Nielsen and Andersen, 1991a). Thus, at d 14 after a single oral dose of CH3HgCI, the relative kidney deposition in female mice was less than half that in male mice (Fig. 11). However, when expressed as amount of mercury deposited in each organ, this difference is almost eliminated due to the higher whole-body retention in female mice than in male mice. However, renal deposition of mercury in male mice was more than fourfold higher than in female mice at d 3, and remained higher throughout the experimental period. Moreover, during an experimental period of 30 d, male mice had significantly lower levels of mercury in blood, brain, liver, and muscles than had female mice (Table 5). Previous studies on sex-related differences in mercury disposition after methylmercury exposure focused exclusively on concentrations in various internal organs. The sex-related differences in relative deposition of mercury in specific organs are toxicologically significant, but when expressed in absolute amounts of mercury the differences are relatively small and do not at all explain the large difference in the whole-body retention between males and females previously reported in rats (Thomas et al., 1986) and mice (Nielsen and Andersen, 1990). Accordingly, it was recently demonstrated that the main cause for a sexual difference in mercury disposition observed after more than a

). B. NIELSEN

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TABLE 5. Mercury in Whole-Body, Carcass, Blood, Hair, Dermis, Abdominal Fat, Femoral Muscle, Thigh Bone, Liver, Kidney, and Brain of Male and Female Borrr.NMRI Mice (n - 10 per group) at Increasing Periods After a Single Oral Dose of Methylmercuric Chloride (1 ^mol/kg) Location

Sex

d3

d 10

d20

WBR WBR

F M

32.2 (31.6-35.1) 38.2 (34.7-39.9)

21.9 (21.3-23.5) 18.2 (12.8-22.9)

12.3 (12.1-14.1) 6.2 (3.0-7.4)

7.3 (6.5-8.6) 4.1 (3.6-4.5)

Carcass Carcass

F M

18.8 (18.3-20.1) 19.9 (19.2-21.2)

14.8 (13.3-15.0) 10.6 (9.5-10.8)

8.3 (7.8-8.8) 3.5 (3.2-3.7)

6.0 (5.7-6.3) 2.8 (2.8-3.1)

Blood Blood

F M

656 (499-842) 382 (294-542)

334 (240-436) 193 (105-241)

161 (145-188) 68 (54-85)

Hair Hair

F M

40 (32-61) 118 (100-243)

245 (75-6845) 280 (118-1574)

Dermis Dermis

F M

Fat Fat

F M

Muscle Muscle

F M

1039 (957-1215) 766 (710-895)

Bone Bone

F M

b.d. b.d.

b.d. b.d.

b.d. b.d.

Liver Liver

F M

3070 (2616-3549) 3185 (2870-3553)

2018 (1811-2097) 1628 (1274-2107)

806 (685-904) 531 (282-700)

382 (309-510) 164 (127-339)

Kidney Kidney

F M

1652 (1407-1773) 7361 (5807-8073)

1210 (1096-1290) 3004 (2052-3790)

595 (555-646) 856 (582-1304)

355 (313-371) 464 (245-685)

Brain Brain

F M

237 (211-250) 207 (160-233)

220 (210-230) 159 (117-182)

b.d. 47

(43-56)

455 (381-634) 198 (121-223) 63 75

(53-132) (51-95)

197 (181-221) 98 (72-166) 53 19

(39-77) (18-40)

639 (614-678) 413 (245-494)

d30

87 46

(71-431) (35-111)

22 13

(16-32) (8-19)

293 (238-332) 151 (106-185)

111 53

1911 (459-5469) 1084 (666-1943) 108 (58-240) 48 (31-84) 10 5

(7-11) (2-9)

129 (104-143) 28 (20-62) b.d. b.d.

(96-124) (30-62)

59 (42-65) 25 (18-39)

Nofe. Results are expressed in nmol (whole-body retention and carcass), in pmol (liver, kidney, brain), and in pmol mercury/g and given as medians with 25th and 75th percentiles. From Nielsen and Andersen (1991a). b.d., Below detection limit. WBR, whole-body retention.

week is the difference in carcass deposition (Nielsen and Andersen, 1991a). Thus, the difference between the amounts of mercury deposited in carcasses from male and female mice (Table 5) almost accounts for the difference in whole-body retention. The lower rate of elimination from carcass than from the whole body after methylmercury exposure also explains why the whole-body elimination does not follow firstorder kinetics. However, significant sexual differences do exist in the toxicokinetics of methylmercury, as kidney deposition at d 3 and d 10 is significantly higher in male mice than in female mice (Table 5). More-



115

over, comparison of blood concentrations of mercury with whole-body and carcass levels of mercury (Table 5) illustrates that, despite comparable whole-body retentions and carcass depositions observed in male and female mice, the blood concentration in male mice is only about half the blood concentration in female mice. Furthermore, elimination half-times for mercury in blood are not very different in male and female mice and are close to whole-body half-times in male mice. However, in female mice the half-times of mercury in blood and the whole body are different. As the blood concentration of mercury is larger in females than in males, the difference in toxicokinetics of methylmercury is not likely to be caused by different rates of liver-to-blood transfer, but rather by differences in the blood-to-urine transfer. This would also explain the higher deposition of mercury in brain, muscles, fat, dermis, and hair in females than in males (Table 5). Estimation from blood concentrations of the body burden of mercury in mice is therefore complicated. Thomas et al. (1986) suggested that different deposition of mercury in pelt (integument separated from underlying muscle and fat) could be a major factor in the sex-related difference in toxicokinetics between male and female rats. However, measurements of mercury in pelt by these authors yielded no significant sexual difference between 4 and 32 d after CHjHgCI administration. The majority (85%) of mercury deposited in hair was organic mercury. As whole-body levels of mercury decreased with time while the hair levels remained rather constant, the results demonstrated a marked increase in the relative deposition of mercury in pelt with time, in accordance with an earlier study on relative skin and fur deposition of methylmercury in rats (Magos and Butler, 1976). A study on the influence of sex on retention of mercuric mercury demonstrated significant differences in amounts of mercury deposited in hair at d 9 after intraperitoneal administration of HgCI2 to male and female mice (Kostial et al., 1985). Mercury found in the dermis after shaving is probably deposited in newly grown hair, and the rate of deposition would be expected to reflect the blood levels during the growth period. The reduced dermis levels with time therefore reflect the reduced blood levels of mercury in combination with outgrowth of hair. The incorporation of mercury into hair is an irreversible deposition, as elimination is solely through loss of hair. As the relative amount of mercury in hair increases in relation to the decreasing whole-body retention, the whole-body elimination will deviate from first-order kinetics. Thus, the present study demonstrates that first-order kinetics is observed only during 2-3 wk after CH3HgCI exposure. Comparisons of hair levels of mercury between male and female mice demonstrated higher median concentrations of mercury in male mice than in female mice during the first 3 wk of the experimental period (Table 5). However, at d 30 after CH3HgCI administration, the females


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had about twice as much mercury per gram of hair (median values) as male mice. At d 20 and 30, very large variations in mercury concentration within groups were observed. The large variation in mercury concentration in hair at d 20 and 30 is probably due to the noncontinuous growth of hair. Thus, hair growth in mice is divided into three to four stages (Wolfe and Coleman, 1975). Therefore growth and thereby incorporation of mercury take place only during part of a time period, resulting in very differentiated incorporations of mercury during a limited study period after a single exposure as in the present experiment. Skeletal muscle accounts for almost half the body weight of rats. Accordingly, deposition of even relatively low concentrations in this tissue could represent a major part of mercury retained in the body. Only limited experimental evidence on the muscular deposition has been published so far. In the study of Thomas et al. (1986) mercury deposition in skeletal muscle (from rectis femuris) increased during the first 2 d after subcutaneous administration of CH3HgCI (1 /imol/kg) to rats followed by a decrease throughout the rest of the 98-d experimental period. No sexual difference in muscular deposition was observed (Thomas et al., 1986). The present study (Table 5) points to the muscles as an important deposit for mercury in mice after CH3HgCI (Nielsen and Andersen, 1991a). The toxicokinetics of methylmercury has hitherto been studied only to a limited extent using both whole-body retention data and data on concentrations of mercury in specific organs and tissues in the same experiment. Thus, the results on sex-related differences in the deposition between organs and tissues in Bom:NMRI mice need further investigation and validation by the use of other mice strains, as strain differences in the toxicokinetics of methylmercury have been demonstrated (Nielsen and Andersen, 1991a).

CONCLUSIONS

A valid and accurate method using the gamma-emitting isotope 203Hg for the study of the toxicokinetics of both organic and inorganic mercury has been developed and used to study the toxicokinetics of HgCI2 and CH3HgCI. Mercuric Chloride

1. The fractional whole-body retention of mercury at 14 d after oral or intraperitoneal dosage was inversely related to the dose size, conceivably due to damage to the kidneys resulting in augmented loss of mercury with the urine at the highest dose levels. Moreover, at the


2.

3.


4.

5.

6.

117

highest doses of HgCI2, a delay in fecal elimination of nonabsorbed mercury was observed, indicating a decreased peristaltic rate. An estimated "true absorption" of a single oral dose of HgCI2 was calculated to be about 20% at 2 different dose levels, that is, twice as high as earlier estimates that did not take into account the possible rapid reexcretion of absorbed mercury during the experimental period. The different excretion rates between orally and intraperitoneally administered HgCI2 are most likely due to different dispositions of systemically delivered and retained mercury. The rate of excretion was independent of the dose within the dose range used in these experiments. The relative organ distribution of retained mercury after HgCI2 exposure depended on dose and was characterized by increasing relative deposition in liver, stomach, intestines, testes, spleen, and carcass but decreasing relative renal deposition with increasing dose. Organ distribution of mercury after HgCI2 exposure is different both in terms of relative organ distribution and in quantitative terms due to different whole-body retentions between orally and parenterally administered mice and between male and female mice. The differences in the toxicokinetics of orally and parenterally administered inorganic mercury are probably due to a first-pass effect. Thus, absorbed mercury would be able to bind to minor proteins (i.e., metallothionein, glutathione), which would change the availability for organ deposition, before being circulated to other organs. The toxicokinetics of inorganic mercury in mice depend on dose size, administration route, and sex; the mouse strain is of less importance than the other factors. As human exposure is often via the oral route, a toxicokinetic analysis emerging from an experimental model using this exposure route is of relevance for risk evaluation in relation to human mercury exposure. Methylmercuric Chloride

1. The elimination of mercury was demonstrated to follow first-order kinetics during a 2-wk study period independently of administration route, strain, or sex. The increasing relative carcass deposition with time (slower rate of excretion) explains the observed deviation from first-order elimination kinetics observed during longer experimental periods. 2. As the intestinal absorption of mercury was demonstrated to be almost complete, the observed inverse relationship between administered dose and whole-body retention was not caused by an effect on the intestinal uptake mechanism.


118

] . B. NIELSEN

3. Dose and route of administration of CH3HgCI did not affect the relative organ distribution of mercury significantly. 4. A significant difference in whole-body retention of mercury was observed between different strains of inbred mice at d 14 after administration of CH3HgCI. The relative organ distribution of mercury also varied significantly between different strains of mice. 5. The large sexual differences in mercury levels in tissues compared with the more limited differences in half-times and the fact that several organs have different mercury levels already at d 3 after dosage, although the whole-body levels at d 3 are more or less equal, indicate extensive differences in the methylmercury toxicokinetics between male and female mice. 6. As the blood concentration of mercury is larger in females than in males, the difference in toxicokinetics of methylmercury is not likely to be caused by different rates of liver-to-blood transfer, but rather by differences in the blood to urine transfer. This would also explain the higher deposition of mercury in brain, muscles, fat, dermis, and hair in females than in males. Estimation of the body burden of mercury from blood concentrations is therefore complicated. 7. The results on sex-related differences in the deposition between organs and tissues in Bom:NMRI mice need further investigation and validation by the use of other mice strains as well, as strain differences in the toxicokinetics of methylmercury have been demonstrated. Suggestions for Further Studies

Despite increasing knowledge of the toxicokinetics of mercuric mercury and methylmercury, the experimental basis for a risk assessment in relation to human exposure to mercuric compounds could still be improved through further studies. Besides confirmation of results obtained in some earlier studies, the following questions or problems seem relevant to study. 1. Most toxicokinetic studies of mercuric compounds have used either single-dose administration or short-term exposures, despite the fact that most humans are chronically or subchronically exposed. An investigation of the agreement between the toxicokinetics of mercuric compounds after a single dose and after chronic or subchronic exposure would therefore be relevant. 2. Most earlier studies used doses of mercury much higher than the human exposure situation. The influence of dose on gastrointestinal absorption and organ deposition of inorganic mercury at very low exposures would therefore by interesting. 3. A decreased peristaltic rate would cause the orally ingested inorganic mercury to stay for a longer period in the intestinal tract and perhaps


4. 5.

6.


7.

8.

119

thereby cause an increased fractional absorption. A closer investigation of the effect of dose of inorganic mercury on the peristaltic rate could be investigated by using the fecal marker 51CrCI3. No in vivo studies have yet been published on the actual sites of absorption of mercuric compounds in the intestinal tract. Identification of the possible biochemical background for the observed difference in methylmercury excretion between males and females is relevant, as present human risk assessment does not differentiate between males and females. Hair is a major deposit for mercury after exposure to methylmercury. An investigation of the toxicokinetics of methylmercury in hairless mice might therefore disclose different toxicokinetics that are perhaps of more relevance to humans. The present levels of human exposure to mercuric compounds would normally not be expected to cause adverse toxicity to adults. However, fetuses and children might be prominent targets also at low exposures. Therefore, animal studies of exposure to mercury through placenta or mothers' milk are relevant. In relation to these studies, relevant factors include the influence of diet and concomitant exposure to other compounds (i.e., selenium, alcohol), which could possibly affect the transport or organ deposition of absorbed mercury. Most human and experimental studies that investigate deposition, excretion, or other toxicokinetic parameters do not speciate the mercury. In this context, speciation is not only a matter of organic or inorganic mercury, but also identification of the specific lowmolecular-weight compounds and proteins that bind the mercuric compounds. REFERENCES

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Studies on the dominant-lethal and fertility effects of the heavy metal compounds methylmercuric hydroxide, mercuric chloride, and cadmium chloride in male and female mice.

Mercuric chloride-induced autoimmunity.

An electrophysiological study of the action of methylmercuric chloride and mercuric chloride on the sciatic nerve-sartorius muscle preparation of the frog.

Mercuric chloride inhibition of leucocyte migration.

Screening of common bacteria capable of demethylation of methylmercuric chloride.

Transformation of mercuric chloride and methylmercury by the rumen microflora.

Disposition and retention of mercuric chloride in mice after oral and parenteral administration.

Mercuric chloride-induced kidney damage in mice: time course and effect of dose.

Chronic mercuric chloride intoxication in digestive system of Channa punctatus.

Mercuric chloride-induced renal tubular necrosis in the rat.

Epidermal growth factor accelerates renal repair in mercuric chloride nephrotoxicity.

Leucocyte aggregation and lymphocyte transformation induced by mercuric chloride.

Alanine aminopeptidase excretion after mercuric chloride renal failure.

Plasmid conferring increased sensitivity to mercuric chloride and cobalt chloride found in some laboratory strains of Escherichia coli K-12.

The effects of dimercaptosuccinic acid on the excretion and distribution of mercury in rats and mice treated with mercuric chloride and methylmercury chloride.

Glutathione monoethyl ester moderates mercuric chloride-induced acute renal failure.

Microautoradiographic study of the distribution of methylmercuric (203) chloride in the Mallard duck (Anas platyrhynchos).

Lactating and nonlactating rats differ to renal toxicity induced by mercuric chloride: the preventive effect of zinc chloride.

Metabolism of methylmercuric chloride by the gastro-intestinal flora of the rat.

Alterations in gene expression after chronic treatment of glioma cells in culture with methylmercuric chloride.

Rate of demethylation of methylmercuric chloride by Enterobacter aerogenes and Serratia marcescens.

Inhibition of implantation caused by methylmercury and mercuric chloride in mouse embryos in vivo.

Synthesis and evaluation of sulfur-containing steroids against methylmercuric chloride toxicity.

Increased urinary excretion of zinc and copper by mercuric chloride injection in rats.