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Metabolism of ethylmercuric chloride-203Hg in rats
TOXICOLOGYANDAPPLIEDPHARMACOLOGY17,181-188(1970)
Metabolism
of Ethylmercuric
Chloride=O’Hg
in Rats*
YASUSHI TAKEDA AND TYUNOSIN UKITA Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Received August 13,1969
Metabolism of Ethylmercuric Chloride-203Hgin Rats. TAKEDA, YASUSHI, and UKITA, TYUNOSIN (1970). Toxicol. Appl. Pharmacol. 17, 181-188. Methods for the analysisof the mercury compoundsin biological samples were establishedand applied to the investigation of the metabolic fate of ethylmercuric chloride 203Hgin rats. Analysesof mercury compoundscontained in the liver, kidney, urine, and fecesof rats treatedwith ethylmercuric chloride-203Hg(20 mg Hg/kg, SC)indicated that the content of organic mercury in the kidney washigherthan in the liver, that the former increased with time, and that the organic mercury content in feceswashigherthan in the urine. The organic mercury compoundin theseorgansand excreta was identified as the ethylmercury derivative, and mercury metabolitesother than inorganicmercury were not detected.The mercury compoundseemed to be bound to protein in the organs.
In our previous papers it was pointed out that when alkyl-mercury compounds were administered to rats, the accumulation of mercury in the blood, liver, kidney, and muscle was quite marked and long-lasting (Takeda et al., 1968a), and that, in the blood of rats treated with ethylmercuric chloride, ethylmercury residues were found to be bound to SH groups of hemoglobin (Takeda et al., 1968b). There have been several reports (Miller et al., 1961; Gage, 1964; Sadakane, 1964) on the fate of the alkylmercury compound in the animal body. These, however, did not deal with the identification of the mercury compounds accumulating in the body. In order to have more detailed knowledge on the metabolism of alkylmercury compounds, methods for the identification of the mercury compounds in biological samples were developed and applied to the studies on the fate of ethylmercuric chloride in rats; the results obtained are the subject of the present communication. METHODS 203Hg-labeled ethylmercuric chloride (EMC-203Hg) was prepared from (N03)2203Hg as described previously (Takeda et al., 1968a); the specific activity was ca. 20,000 counts/min/pg Hg. Determination of radioactivity
was carried out with a well-type NaI(T1) crystal
scintillation counter, and radioactive scannings of thin-layer chromatograms formed with a Packard Radio-chromatogram Scanner System, Model 7201.
were per-
* Presented in part at the 88thAnnual Meetingof Pharmaceutical Societyof Japan,Tokyo, April 1968. 181
182
TAKEDA
AND
UKITA
Animal experiments. EMC- ‘03Hg dissolved in olive oil was given by subcutaneous injection (20 mg Hg/kg) to male Donryu rats (6 months old; 273-305 g). Individual rats were housed in stainless steel metabolism cages; the diet and water were available at all times. Each day during the experiment, urine and feces were separately sampled and stored in the frozen state (-20”). Two or 8 days after administration, the animals were anesthetized with sodium pentobarbital and killed by bleeding from the carotid. The liver and kidneys were immediately removed, frozen, and stored at -20” until analysis. Analysis of mercury compounds.(a) Organs. To 2 ml of 10 % homogenate of the organ was added successively 0.5 ml of 5 N HCl and 5 ml of 10m3 M dithizone solution in
I
dithiz
inorganic m ercurv dithizonate ; ; ’ I
.J
nate
i
10000
1.0:
.lil 0
OD
: bj
iI
: 5
i
+.< __.._ ,.-I-;t.-’ IO
15
L.* ...‘:..-., 20
25
(ml)
FIG. 1. Separation on an alumina column of organic and inorganic mercury dithizonates from the extract of the kidney of a rat administered with ethylmercuric chloride-Z03Hg. The dithizone-CCL, extract (2 ml) and carrier solution (1 ml) (for details, see text) was applied on the column of alumina (activity grade III, 0.5 cm x 3.0 cm) and the column was eluted with Ccl, (10 ml), CC&CHC& (95 : 5) (5 ml), and CC&CHClr (1: 1) (10 ml), successively. Fractions (2 ml) were collected and assayed spectrophotometrically at 470 mp (to estimate the carrier mercury dithizonate) and by measurement of radioactivity (to estimate the 203Hg-labeled mercury dithizonates). The abscissa indicates elution volume (ml), and the ordinate optical density at 470 nm (x - - - - - x) and radioactivity (cpm, -).
Ccl,; the mixture was homogenized in a Waring Blendor. The homogenate was centrifuged (3000 rpm, 20 min); the Ccl, layer was separated and filtered through a dried cotton plug. To 2 ml of the filtrate, a solution ofmercuric and ethylmercuric dithizonates (40 pg Hg each in 1 ml CC14) was added as carrier. The filtrate was applied on an alumina column (activity grade III, 0.5 cm x 3.0 cm), and elution was performed with CCL,, Ccl,-CHCl, (95 : 5), and CCL,-CHCl, (1: l), successively as described by Ishikura and Yokota (1963). A typical elution pattern is shown in Fig. 1. The radioactivity of the organic and inorganic mercury dithizonate fractions was then determined; the percentages for organic and inorganic mercury were calculated from the total.
METABOLISM
OF ETHYLMERCURIC
CHLORIDE
183
The recoveries in the dithizone extraction and in the alumina column chromatography were 99.4 and 95.4 ‘A, respectively. A mercury exchange reaction (Cross and Pinajian, 1951) between the mercury compound extracted from organs and the mercury compounds added as the carrier was not detected. (b) Excreta. Urine (2 ml) was added to 0.5 ml of 5 N HCl and the mixture was extracted with 5 ml of dithizone-CC14. The feces (0.3 g) were homogenized with 5 ml of 1 N HCl, and the homogenate was extracted with 5 ml of dithizone-CC14. The extracts were analyzed as described above. Characterization of organic mercury compounds in the biological samples. Mercury compounds in organs or excreta were extracted with dithizone dissolved in CC& as described above. The extract was applied on an alumina column without the prior addition of carrier in order to eliminate the possibility of an exchange reaction between the mercury contained in the metabolite and that in the carrier. The column was eluted as described above. The fractions obtained by elution with CC14-CHC13 (95: 5), which contained organic mercury dithizonates, were combined, and extracted 3 times each with equal volumes of 0.1% silver acetate solution in 0.1 N acetic acid. The aqueous layer was combined, filtered through a wet cotton plug and added to 0.1 volume of cont. HCl. The solution was extracted 3 times each with equal volumes of CHCl, ; the CHCl, extracts were combined and concentrated in vacua below 30”. The concentrated extracts were applied on thin-layer plates of microcrystalline cellulose2; and the plates were developed in solvent A, isopropanol-conc. ammonia-water (7: 1: 2) and solvent B, n-butanol-ethanol-conc. ammonia (8 : 1: 3). The recovery of the mercury compounds from the organic mercury dithizonate fraction eluted from the column by the final CHCl,-extraction was 71.6 %. Enzymatic digestion of organs. To the 10 % homogenate of liver or kidney was added an equal volume of 0.2 M phosphate buffer, pH 7.0, and 0.2 volume of solution of Pronase-P3 (20 mg/ml),.and the mixture was incubated at 37”. At suitable time intervals aliquots were withdrawn and added to 0.1 volume of 4.4 N perchloric acid. After cooling with ice-water, the mixture was centrifuged. The radioactivity and the ninhydrin coloration (Yemm and Cocking, 1955) of the supernatant were determined. RESULTS Analysis of Mercury
Compounds in Organs and in Excreta
Rats were injected subcutaneously with 203Hg-EMC and the livers and kidneys were removed after 2 or 8 days. Urine and feces were separately collected every 24 hr for 8 days. Mercury compounds in these organs and excreta were extracted with a solution of dithizone in CC4 as mercury dithizonates, and the mercury dithizonates formed were fractionated into organic and inorganic mercury dithizonates by column chromatography on alumina. The results obtained in the organs and excreta are shown in Tables 1 and 2, respectively. In the case of liver, 2 and 8 days after administration the content of the inorganic mercury compound was less than 10 y0 of the total mercury compound in this organ. In the case of kidney, the content of the inorganic mercury was higher than in the liver, and 2 Avicell, Asahi Kasai Co., Ltd., Tokyo, Japan. 3 Kaken Chemical Co., Ltd., Komagome, Bunkyo-ku, Tokyo, Japan.
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TAKEDA
AND
UKITA
furthermore, on day 8 an increase of inorganic mercury compound to approximately twice that seen on day 2 was observed. In the urine, the content of inorganic mercury compound was higher than that of organic mercury compound, but in the feces the content of inorganic mercury compound was much lower than that of organic mercury compound. TABLE 1 ANALYSIS
OF MERCURY COMPOUNDS IN THE ORGANS OF RATS TREATED WITH ETHYLMERCURIC CHLoRIDE-203Hg (20 mg Hg/kg, SC)
Mercury contenta Organs
Days after administration
Liver
2 8
a Each value represents * Organic and inorganic
Organic mercury* (%>
26.2 (23.0-28.8) 30.1 (26.6-34.6) 79.3 (61.7-88.5) 108.7 (97.5-l 15.1)
2 8
Kidney
Total mercury (PPm>
the mean mercury
(range) of samples from are expressed in percent
93.7 (92.4-94.7) 93.8 (93.1-94.2) 82.5 (80.1-85.7) 65.9 (59.5-71.8) three rats. of the total mercury
Inorganic mercury* (%I (5.ty7.6) (5.87:9) 17.5 (14.3-19.9) 34.1 (28.240.5) in the
organ.
Identification of Organic Mercury Compound
The organic mercury dithizonate separated from the organs or excreta as described above was treated successively with silver acetate in dilute acetic acid and HCI to convert the mercury dithizonate to the corresponding organic mercury chloride. Figure 2 shows the thin layer chromatograms of the organic mercury compound extracted from the kidney obtained 2 days after administration of EMC-203Hg. The Rf value of the mercury compound thus extracted from the kidney agreed closely with those obtained for authentic ethylmercuric chloride (Slotta and Jacobi, 1929). The organic mercury compounds obtained from all of the organs and excreta tested revealed a similar behavior in the chromatography systems. Mercury Compoundsin the Liver and Kidney
Two or 8 days after administration of EMC-203Hg, the liver and kidney were taken from the animals, and these organs were homogenized, fractionated into acid-soluble, lipid, nucleic acid, and protein fractions according to the method of Tyner et al. (1953). The distribution of mercury in these fractions is estimated and summarized in Table 3. About 70 % of the mercury accumulated in these organs was found in the protein fraction. Homogenates of these organs were incubated with Pronase, and the increase of mercury content and ninhydrin coloration of acid-soluble fractions was determined. Results for the organ contents 2 days after administration are shown in Fig. 3. In the acid-soluble fraction, the increase in mercury content was found to parallel the increase in ninhydrin coloration. Similar results were obtained on organs sampled 8 days after treatment.
mercury’
(%)
a Each value represents the mean * Total mercury excreted in urine c Organic and inorganic mercury
Feces inorganic
Feces organic mercury’
(“A
mercuryc (%)
Total mercury* (“4
Urine inorganic
2
0.17 (0.02-0.38) 81.9 (79.1-87.1) 18.1 (12.9-20.9)
OF RATS
0.85 (0.44-1.07) 69.5 (63.8-79.2) 30.5 (20.8-36.2)
0.73 (1.54-0.86) 11.6 (8.5-16.1) 88.4 (83.9-91.5)
3
(20 mg H&s,
IN EXCRETA
0.41 (0.18-0.79) 11.3 (8.8-13.8) 88.7 (86.2-91.2)
COMPOUNDS
0.97 (0.83-1.25) 66.8 (59.5-71.1) 33.2 (28.9-40.5)
(332z2.1) 55.6 (37.9-66.6)
1.65 (1.41-2.02)
4
Mercury
SC)
TREATED
2
0.88 (0.83495) 67.7 (65.5-70.4) 32.3 (29.6-34.5)
(40%4.3) 49.4 (35.7-59.3)
1.87 (1.62-2.21)
5
6
67.4 (65.5-69.2) 32.6 (30.8-34.5)
(0.7:?42)
(34%7.8) 56.5 (42.2-66.0)
61.2 (57.7-65.4) 38.8 (34.6-42.3)
(0.7%7)
(35FzO.5) 55.4 (39.5-65.0)
2.11 (1.95-2.29)
7
Cx-fLoRrDE-203Hg
1.74 (1.49-1.98)
ETHYLMERCURIC
content’
WITH
(range) of samples from three rats. Days = days after administration. or feces in each 24-hr period is expressed in percent of the administered dose of mercury. are expressed in percent of the total mercury in each 24-hr period specimen.
0.03 (0.02-0.04) 89.0 (84.8-93.7) 11.0 (6.3-15.2)
0.06 (0.040.08) 72.7 (68.0-78.8) 27.3 (21.2-32.0)
1
OF MERCURY
Days”:
Urine organic mercury= (“4
Total mercury* (“A
Compound
ANALYSIS
TABLE
65.0 (59.5-70.8) 35.0 (29.2-40.5)
(0.7k-?29)
(32%5.8) 58.1 (44.2-67.5)
1.95 (1.88-2.00)
8
i
s 5 E g
z 2 t 0 z B 8 3 $ E f
186
TAKEDA
i-Pl,Oli-conc.
AND
UKITA n-BuOH-EtOH-cont.
NH4011-H,O
(7:1:2)
NHfOH
(8:1:X)
FIG. 2. Radioactivity scans of thin-layer chromatograms prepared with the extract of the kidney from a rat treated with ethylmercuric chloride- 203Hg. Extract of the kidney (for details, see text), was applied on thin-layer plates of microcrystalline cellulose,* and the plates were developed in isopropanolcont. NH,OH-Hz0 (7: 1: 2) (A), and n-butanol-ethanol-conc. NH,OH (8: 1: 3) (B). Solid black bars represent the locations of dithizone-colored spots of standard methylmercuric chloride (MMC) and ethylmercuric chloride (EMC). TABLE
3
DISTRIBUTIONOFMERCURYCOMPOUNDSINTHEORGANS WITHETHYLMERCURYCHLORIDE-~~'H~(~~~~
Organ Liver
Days after administration 2 8
Kidney
2 8
Acid-soluble fraction 14.7 (12.3-18.5) 15.1 (15.0-15.4) 14.1 (12.5-15.6) 18.7 (17.5-21.1)
OF RATSTREATED Hg/kg,sc)
Distribution
of mercury (“/,)
Lipid fraction
Nucleic acid fraction
(3Z.6)
(1.20)
(3.ti.8)
(2.ki.4)
(4.ili.2)
(2.ti.9)
(2.z.8)
(2.z.8)
Protein fraction 73.4 (71.9-74.7) 73.8 (72.2-76.2) 71.4 (70.5-71.9) 69.7 (67.5-71.8)
a Distribution of mercury in each fraction is expressed in percent of the total mercury in the organ, and each value represents the mean (range) of samples from three rats. DISCUSSION Analytical methods for determining materials have been described by Miller
organic
mercury
compounds
in biological
et al. (1961) and by Gage (1964), and have been applied by several investigators to the study of the metabolism of organic mercury compounds. By these methods, however, organic mercury compounds which, on extraction under acidic condition, could not be transferred to the organic solvent have been estimated as inorganic mercury compound. To rule out such ambiguity, the extractions in the present study were performed with dithizone-carbon tetrachloride; this resulted in a
METABOLISMOF ETHYLMERCURIC CHLORIDE
0 I-. 0
1
2
3 Tune
thr)
187
I 3
2 Tune
(hr)
FIG. 3. Relation between the course of proteolytic digestion and release of acid soluble mercury compounds from the kidney (A) and the liver (B) of a rat treated with ethylmercuric chlorideJo3Hg. Acid-soluble fraction was submitted to ninhydrin coloration (Yemm and Cocking, 1955) to measure the extent of the digestion (OD 570 mp, x x) and to determine the release of acid soluble mercury compounds by measurement of radioactivity (o - - - - 0). The time of incubation is represented on the abscissa, and the increment of ninhydrin coloration and radioactivity in the acid-soluble fraction on the ordinates. For further details, see text. quantitative extraction of the mercury compounds in biological sample from acidic homogenates (Takeda et al., 1968b). The mercury dithizonates extracted were separated into fractions of organic and inorganic mercury dithizonates by column chromatography on alumina and the content of mercury in these fractions was determined. Organic mercury dithizonate thus separated was decomposed with silver acetate and the mercury compound converted to the chloride salt by addition of hydrochloric acid. Identification of the organic mercury chloride was carried out by thin-layer chromatography on cellulose. This series of procedures has proved to be fairly suitable for the analysis of mercury compounds in the biological specimens (Nishikate et al., 1968). When the liver, kidney, and excreta of the rats treated with EMC-203Hg were analyzed
by the above described procedure, both inorganic and organic mercury dithizonates were detected. The analysis by thin-layer chromatography of the organic mercury compound derived from the organic mercury-dithizonate fraction showed that the ethylmercury derivative was the sole organic mercury compound that accumulated in the organs and excreta. From these results, it was concluded that a part of the ethylmercury compound administered to the rat was metabolized to inorganic mercury derivatives by cleavage of carbon-mercury bond; biotransformation of the alkyl chain of the alkylmercury moiety, as suggested by Suzuki et al. (1963), was not observed. The accumulation of inorganic mercury was greater in the kidney than in the liver when EMC was administered to rats, and the amount of the inorganic mercury in the kidney increased with time. These observations were interpreted to indicate that the transformation of ethylmercuric compound to inorganic mercury compound occurs mainly in the kidney, or that the inorganic mercury, which has a high affinity for the kidney (Takeda et al., 1968a), once produced from EMC in other organs was retained in the kidney. The content of inorganic mercury compound in the liver of the rat treated with EMC
TAKEDA AND UKITA
188
was low as it was in the feces; therefore, the conversion of the alkylmercury compound to inorganic mercury compound was not prominent in the liver. A similar interpretation has been made by Miller et al. (1961). The results shown in Fig. 3 and Table 3 indicate that the mercury compounds accumulating in the organs were bound to protein, as was previously observed in the case of the blood (Takeda et al., 1968b), and the alkylmercury residue is most likely combined with SH-groups of the protein, forming mercaptide linkages. REFERENCES CROSS,J. M., and PINAJIAN, J. J. (1951). Observation on the exchangeof mercury between phenylmercuricchloride and mercuric chloride using mercury 203. J. Am. Pharm. Assoc., Sci. Ed. 40,95-97. GAGE,J. C. (1964).Distribution and excretion of methyl andphenyl mercury salts.Brit. J. Znd. Med. 21,197-202. ISHIKURA,S., and YOKOTA,K. (1963).Analysis of organicmercury. I. Sensitiveanddifferential determination of phenylmercury acetate and inorganic mercury. Chem. Pharm. Bull. (Tokyo)
11,939-942.
MILLER,V. L., KLAVANO,P.A., JESTAD, A. C., andCsoNKA,E. (1961).Absorption, distribution, and excretion of ethylmercuricchloride. Toxicol. Appl. Pharmacol.3,459-468. NISHIKATA,A., TANZAWA,K., TAKEDA,Y., OSAWA,T., and UKITA, T. (1968). Identification and determinationof mercury compoundsin the hair by extraction with acids.EiseiKagaku 14,211-216.
SADAKANE,T. (1964). Studieson the toxicity of mercury. Report 2. Metabolism of ethylmercuricphosphatein animals.SangyoZgaku6,495-502. SLOTTA,K. H., and JACOBI,K. R. (1929).Uber organischeQuecksilbarbasenund ihre Salze. J. Prakt. Chem.120,249-300. SUZUKI,T., MIYAMA, T., andKATSUNUMA, H. (1963).Comparativestudy of bodily distribution of mercury in mice after subcutaneousadministration of methyl, ethyl, and n-propyl mercury acetate.Japan.J. Exptl. Med. 33,277-282. TAKEDA,Y., KUNUGI,T., HOSHINO, O., and UKITA, T. (1968a).Distribution ofinorganic, aryl, and alkyl mercury compoundsin rats. Toxicol. Appl. Pharmacol.13,156-164. TAKEDA,Y., KUNUGI,T., TERAO,T., and UKITA, T. (1968b).Mercury compoundsin the blood of rats treated with ethylmercuric chloride. Toxicol. Appl. Pharmacol.13,165-173. TYNER,E. P., HEIDERBERGER, C., and LEPAGE, G. A. (1953).Intracellular distribution of radioactivity in nucleic acid nucleotidesand proteins following simultaneousadministration of P32and glycine-2-C14.CancerRes.13, 186-203. YEMM, E. W., and COCKING,E. C. (1955). Determination of amino acids with ninhydrin. Analyst 80,209-213.
Metabolism
of Ethylmercuric
Chloride=O’Hg
in Rats*
YASUSHI TAKEDA AND TYUNOSIN UKITA Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Received August 13,1969
Metabolism of Ethylmercuric Chloride-203Hgin Rats. TAKEDA, YASUSHI, and UKITA, TYUNOSIN (1970). Toxicol. Appl. Pharmacol. 17, 181-188. Methods for the analysisof the mercury compoundsin biological samples were establishedand applied to the investigation of the metabolic fate of ethylmercuric chloride 203Hgin rats. Analysesof mercury compoundscontained in the liver, kidney, urine, and fecesof rats treatedwith ethylmercuric chloride-203Hg(20 mg Hg/kg, SC)indicated that the content of organic mercury in the kidney washigherthan in the liver, that the former increased with time, and that the organic mercury content in feceswashigherthan in the urine. The organic mercury compoundin theseorgansand excreta was identified as the ethylmercury derivative, and mercury metabolitesother than inorganicmercury were not detected.The mercury compoundseemed to be bound to protein in the organs.
In our previous papers it was pointed out that when alkyl-mercury compounds were administered to rats, the accumulation of mercury in the blood, liver, kidney, and muscle was quite marked and long-lasting (Takeda et al., 1968a), and that, in the blood of rats treated with ethylmercuric chloride, ethylmercury residues were found to be bound to SH groups of hemoglobin (Takeda et al., 1968b). There have been several reports (Miller et al., 1961; Gage, 1964; Sadakane, 1964) on the fate of the alkylmercury compound in the animal body. These, however, did not deal with the identification of the mercury compounds accumulating in the body. In order to have more detailed knowledge on the metabolism of alkylmercury compounds, methods for the identification of the mercury compounds in biological samples were developed and applied to the studies on the fate of ethylmercuric chloride in rats; the results obtained are the subject of the present communication. METHODS 203Hg-labeled ethylmercuric chloride (EMC-203Hg) was prepared from (N03)2203Hg as described previously (Takeda et al., 1968a); the specific activity was ca. 20,000 counts/min/pg Hg. Determination of radioactivity
was carried out with a well-type NaI(T1) crystal
scintillation counter, and radioactive scannings of thin-layer chromatograms formed with a Packard Radio-chromatogram Scanner System, Model 7201.
were per-
* Presented in part at the 88thAnnual Meetingof Pharmaceutical Societyof Japan,Tokyo, April 1968. 181
182
TAKEDA
AND
UKITA
Animal experiments. EMC- ‘03Hg dissolved in olive oil was given by subcutaneous injection (20 mg Hg/kg) to male Donryu rats (6 months old; 273-305 g). Individual rats were housed in stainless steel metabolism cages; the diet and water were available at all times. Each day during the experiment, urine and feces were separately sampled and stored in the frozen state (-20”). Two or 8 days after administration, the animals were anesthetized with sodium pentobarbital and killed by bleeding from the carotid. The liver and kidneys were immediately removed, frozen, and stored at -20” until analysis. Analysis of mercury compounds.(a) Organs. To 2 ml of 10 % homogenate of the organ was added successively 0.5 ml of 5 N HCl and 5 ml of 10m3 M dithizone solution in
I
dithiz
inorganic m ercurv dithizonate ; ; ’ I
.J
nate
i
10000
1.0:
.lil 0
OD
: bj
iI
: 5
i
+.< __.._ ,.-I-;t.-’ IO
15
L.* ...‘:..-., 20
25
(ml)
FIG. 1. Separation on an alumina column of organic and inorganic mercury dithizonates from the extract of the kidney of a rat administered with ethylmercuric chloride-Z03Hg. The dithizone-CCL, extract (2 ml) and carrier solution (1 ml) (for details, see text) was applied on the column of alumina (activity grade III, 0.5 cm x 3.0 cm) and the column was eluted with Ccl, (10 ml), CC&CHC& (95 : 5) (5 ml), and CC&CHClr (1: 1) (10 ml), successively. Fractions (2 ml) were collected and assayed spectrophotometrically at 470 mp (to estimate the carrier mercury dithizonate) and by measurement of radioactivity (to estimate the 203Hg-labeled mercury dithizonates). The abscissa indicates elution volume (ml), and the ordinate optical density at 470 nm (x - - - - - x) and radioactivity (cpm, -).
Ccl,; the mixture was homogenized in a Waring Blendor. The homogenate was centrifuged (3000 rpm, 20 min); the Ccl, layer was separated and filtered through a dried cotton plug. To 2 ml of the filtrate, a solution ofmercuric and ethylmercuric dithizonates (40 pg Hg each in 1 ml CC14) was added as carrier. The filtrate was applied on an alumina column (activity grade III, 0.5 cm x 3.0 cm), and elution was performed with CCL,, Ccl,-CHCl, (95 : 5), and CCL,-CHCl, (1: l), successively as described by Ishikura and Yokota (1963). A typical elution pattern is shown in Fig. 1. The radioactivity of the organic and inorganic mercury dithizonate fractions was then determined; the percentages for organic and inorganic mercury were calculated from the total.
METABOLISM
OF ETHYLMERCURIC
CHLORIDE
183
The recoveries in the dithizone extraction and in the alumina column chromatography were 99.4 and 95.4 ‘A, respectively. A mercury exchange reaction (Cross and Pinajian, 1951) between the mercury compound extracted from organs and the mercury compounds added as the carrier was not detected. (b) Excreta. Urine (2 ml) was added to 0.5 ml of 5 N HCl and the mixture was extracted with 5 ml of dithizone-CC14. The feces (0.3 g) were homogenized with 5 ml of 1 N HCl, and the homogenate was extracted with 5 ml of dithizone-CC14. The extracts were analyzed as described above. Characterization of organic mercury compounds in the biological samples. Mercury compounds in organs or excreta were extracted with dithizone dissolved in CC& as described above. The extract was applied on an alumina column without the prior addition of carrier in order to eliminate the possibility of an exchange reaction between the mercury contained in the metabolite and that in the carrier. The column was eluted as described above. The fractions obtained by elution with CC14-CHC13 (95: 5), which contained organic mercury dithizonates, were combined, and extracted 3 times each with equal volumes of 0.1% silver acetate solution in 0.1 N acetic acid. The aqueous layer was combined, filtered through a wet cotton plug and added to 0.1 volume of cont. HCl. The solution was extracted 3 times each with equal volumes of CHCl, ; the CHCl, extracts were combined and concentrated in vacua below 30”. The concentrated extracts were applied on thin-layer plates of microcrystalline cellulose2; and the plates were developed in solvent A, isopropanol-conc. ammonia-water (7: 1: 2) and solvent B, n-butanol-ethanol-conc. ammonia (8 : 1: 3). The recovery of the mercury compounds from the organic mercury dithizonate fraction eluted from the column by the final CHCl,-extraction was 71.6 %. Enzymatic digestion of organs. To the 10 % homogenate of liver or kidney was added an equal volume of 0.2 M phosphate buffer, pH 7.0, and 0.2 volume of solution of Pronase-P3 (20 mg/ml),.and the mixture was incubated at 37”. At suitable time intervals aliquots were withdrawn and added to 0.1 volume of 4.4 N perchloric acid. After cooling with ice-water, the mixture was centrifuged. The radioactivity and the ninhydrin coloration (Yemm and Cocking, 1955) of the supernatant were determined. RESULTS Analysis of Mercury
Compounds in Organs and in Excreta
Rats were injected subcutaneously with 203Hg-EMC and the livers and kidneys were removed after 2 or 8 days. Urine and feces were separately collected every 24 hr for 8 days. Mercury compounds in these organs and excreta were extracted with a solution of dithizone in CC4 as mercury dithizonates, and the mercury dithizonates formed were fractionated into organic and inorganic mercury dithizonates by column chromatography on alumina. The results obtained in the organs and excreta are shown in Tables 1 and 2, respectively. In the case of liver, 2 and 8 days after administration the content of the inorganic mercury compound was less than 10 y0 of the total mercury compound in this organ. In the case of kidney, the content of the inorganic mercury was higher than in the liver, and 2 Avicell, Asahi Kasai Co., Ltd., Tokyo, Japan. 3 Kaken Chemical Co., Ltd., Komagome, Bunkyo-ku, Tokyo, Japan.
184
TAKEDA
AND
UKITA
furthermore, on day 8 an increase of inorganic mercury compound to approximately twice that seen on day 2 was observed. In the urine, the content of inorganic mercury compound was higher than that of organic mercury compound, but in the feces the content of inorganic mercury compound was much lower than that of organic mercury compound. TABLE 1 ANALYSIS
OF MERCURY COMPOUNDS IN THE ORGANS OF RATS TREATED WITH ETHYLMERCURIC CHLoRIDE-203Hg (20 mg Hg/kg, SC)
Mercury contenta Organs
Days after administration
Liver
2 8
a Each value represents * Organic and inorganic
Organic mercury* (%>
26.2 (23.0-28.8) 30.1 (26.6-34.6) 79.3 (61.7-88.5) 108.7 (97.5-l 15.1)
2 8
Kidney
Total mercury (PPm>
the mean mercury
(range) of samples from are expressed in percent
93.7 (92.4-94.7) 93.8 (93.1-94.2) 82.5 (80.1-85.7) 65.9 (59.5-71.8) three rats. of the total mercury
Inorganic mercury* (%I (5.ty7.6) (5.87:9) 17.5 (14.3-19.9) 34.1 (28.240.5) in the
organ.
Identification of Organic Mercury Compound
The organic mercury dithizonate separated from the organs or excreta as described above was treated successively with silver acetate in dilute acetic acid and HCI to convert the mercury dithizonate to the corresponding organic mercury chloride. Figure 2 shows the thin layer chromatograms of the organic mercury compound extracted from the kidney obtained 2 days after administration of EMC-203Hg. The Rf value of the mercury compound thus extracted from the kidney agreed closely with those obtained for authentic ethylmercuric chloride (Slotta and Jacobi, 1929). The organic mercury compounds obtained from all of the organs and excreta tested revealed a similar behavior in the chromatography systems. Mercury Compoundsin the Liver and Kidney
Two or 8 days after administration of EMC-203Hg, the liver and kidney were taken from the animals, and these organs were homogenized, fractionated into acid-soluble, lipid, nucleic acid, and protein fractions according to the method of Tyner et al. (1953). The distribution of mercury in these fractions is estimated and summarized in Table 3. About 70 % of the mercury accumulated in these organs was found in the protein fraction. Homogenates of these organs were incubated with Pronase, and the increase of mercury content and ninhydrin coloration of acid-soluble fractions was determined. Results for the organ contents 2 days after administration are shown in Fig. 3. In the acid-soluble fraction, the increase in mercury content was found to parallel the increase in ninhydrin coloration. Similar results were obtained on organs sampled 8 days after treatment.
mercury’
(%)
a Each value represents the mean * Total mercury excreted in urine c Organic and inorganic mercury
Feces inorganic
Feces organic mercury’
(“A
mercuryc (%)
Total mercury* (“4
Urine inorganic
2
0.17 (0.02-0.38) 81.9 (79.1-87.1) 18.1 (12.9-20.9)
OF RATS
0.85 (0.44-1.07) 69.5 (63.8-79.2) 30.5 (20.8-36.2)
0.73 (1.54-0.86) 11.6 (8.5-16.1) 88.4 (83.9-91.5)
3
(20 mg H&s,
IN EXCRETA
0.41 (0.18-0.79) 11.3 (8.8-13.8) 88.7 (86.2-91.2)
COMPOUNDS
0.97 (0.83-1.25) 66.8 (59.5-71.1) 33.2 (28.9-40.5)
(332z2.1) 55.6 (37.9-66.6)
1.65 (1.41-2.02)
4
Mercury
SC)
TREATED
2
0.88 (0.83495) 67.7 (65.5-70.4) 32.3 (29.6-34.5)
(40%4.3) 49.4 (35.7-59.3)
1.87 (1.62-2.21)
5
6
67.4 (65.5-69.2) 32.6 (30.8-34.5)
(0.7:?42)
(34%7.8) 56.5 (42.2-66.0)
61.2 (57.7-65.4) 38.8 (34.6-42.3)
(0.7%7)
(35FzO.5) 55.4 (39.5-65.0)
2.11 (1.95-2.29)
7
Cx-fLoRrDE-203Hg
1.74 (1.49-1.98)
ETHYLMERCURIC
content’
WITH
(range) of samples from three rats. Days = days after administration. or feces in each 24-hr period is expressed in percent of the administered dose of mercury. are expressed in percent of the total mercury in each 24-hr period specimen.
0.03 (0.02-0.04) 89.0 (84.8-93.7) 11.0 (6.3-15.2)
0.06 (0.040.08) 72.7 (68.0-78.8) 27.3 (21.2-32.0)
1
OF MERCURY
Days”:
Urine organic mercury= (“4
Total mercury* (“A
Compound
ANALYSIS
TABLE
65.0 (59.5-70.8) 35.0 (29.2-40.5)
(0.7k-?29)
(32%5.8) 58.1 (44.2-67.5)
1.95 (1.88-2.00)
8
i
s 5 E g
z 2 t 0 z B 8 3 $ E f
186
TAKEDA
i-Pl,Oli-conc.
AND
UKITA n-BuOH-EtOH-cont.
NH4011-H,O
(7:1:2)
NHfOH
(8:1:X)
FIG. 2. Radioactivity scans of thin-layer chromatograms prepared with the extract of the kidney from a rat treated with ethylmercuric chloride- 203Hg. Extract of the kidney (for details, see text), was applied on thin-layer plates of microcrystalline cellulose,* and the plates were developed in isopropanolcont. NH,OH-Hz0 (7: 1: 2) (A), and n-butanol-ethanol-conc. NH,OH (8: 1: 3) (B). Solid black bars represent the locations of dithizone-colored spots of standard methylmercuric chloride (MMC) and ethylmercuric chloride (EMC). TABLE
3
DISTRIBUTIONOFMERCURYCOMPOUNDSINTHEORGANS WITHETHYLMERCURYCHLORIDE-~~'H~(~~~~
Organ Liver
Days after administration 2 8
Kidney
2 8
Acid-soluble fraction 14.7 (12.3-18.5) 15.1 (15.0-15.4) 14.1 (12.5-15.6) 18.7 (17.5-21.1)
OF RATSTREATED Hg/kg,sc)
Distribution
of mercury (“/,)
Lipid fraction
Nucleic acid fraction
(3Z.6)
(1.20)
(3.ti.8)
(2.ki.4)
(4.ili.2)
(2.ti.9)
(2.z.8)
(2.z.8)
Protein fraction 73.4 (71.9-74.7) 73.8 (72.2-76.2) 71.4 (70.5-71.9) 69.7 (67.5-71.8)
a Distribution of mercury in each fraction is expressed in percent of the total mercury in the organ, and each value represents the mean (range) of samples from three rats. DISCUSSION Analytical methods for determining materials have been described by Miller
organic
mercury
compounds
in biological
et al. (1961) and by Gage (1964), and have been applied by several investigators to the study of the metabolism of organic mercury compounds. By these methods, however, organic mercury compounds which, on extraction under acidic condition, could not be transferred to the organic solvent have been estimated as inorganic mercury compound. To rule out such ambiguity, the extractions in the present study were performed with dithizone-carbon tetrachloride; this resulted in a
METABOLISMOF ETHYLMERCURIC CHLORIDE
0 I-. 0
1
2
3 Tune
thr)
187
I 3
2 Tune
(hr)
FIG. 3. Relation between the course of proteolytic digestion and release of acid soluble mercury compounds from the kidney (A) and the liver (B) of a rat treated with ethylmercuric chlorideJo3Hg. Acid-soluble fraction was submitted to ninhydrin coloration (Yemm and Cocking, 1955) to measure the extent of the digestion (OD 570 mp, x x) and to determine the release of acid soluble mercury compounds by measurement of radioactivity (o - - - - 0). The time of incubation is represented on the abscissa, and the increment of ninhydrin coloration and radioactivity in the acid-soluble fraction on the ordinates. For further details, see text. quantitative extraction of the mercury compounds in biological sample from acidic homogenates (Takeda et al., 1968b). The mercury dithizonates extracted were separated into fractions of organic and inorganic mercury dithizonates by column chromatography on alumina and the content of mercury in these fractions was determined. Organic mercury dithizonate thus separated was decomposed with silver acetate and the mercury compound converted to the chloride salt by addition of hydrochloric acid. Identification of the organic mercury chloride was carried out by thin-layer chromatography on cellulose. This series of procedures has proved to be fairly suitable for the analysis of mercury compounds in the biological specimens (Nishikate et al., 1968). When the liver, kidney, and excreta of the rats treated with EMC-203Hg were analyzed
by the above described procedure, both inorganic and organic mercury dithizonates were detected. The analysis by thin-layer chromatography of the organic mercury compound derived from the organic mercury-dithizonate fraction showed that the ethylmercury derivative was the sole organic mercury compound that accumulated in the organs and excreta. From these results, it was concluded that a part of the ethylmercury compound administered to the rat was metabolized to inorganic mercury derivatives by cleavage of carbon-mercury bond; biotransformation of the alkyl chain of the alkylmercury moiety, as suggested by Suzuki et al. (1963), was not observed. The accumulation of inorganic mercury was greater in the kidney than in the liver when EMC was administered to rats, and the amount of the inorganic mercury in the kidney increased with time. These observations were interpreted to indicate that the transformation of ethylmercuric compound to inorganic mercury compound occurs mainly in the kidney, or that the inorganic mercury, which has a high affinity for the kidney (Takeda et al., 1968a), once produced from EMC in other organs was retained in the kidney. The content of inorganic mercury compound in the liver of the rat treated with EMC
TAKEDA AND UKITA
188
was low as it was in the feces; therefore, the conversion of the alkylmercury compound to inorganic mercury compound was not prominent in the liver. A similar interpretation has been made by Miller et al. (1961). The results shown in Fig. 3 and Table 3 indicate that the mercury compounds accumulating in the organs were bound to protein, as was previously observed in the case of the blood (Takeda et al., 1968b), and the alkylmercury residue is most likely combined with SH-groups of the protein, forming mercaptide linkages. REFERENCES CROSS,J. M., and PINAJIAN, J. J. (1951). Observation on the exchangeof mercury between phenylmercuricchloride and mercuric chloride using mercury 203. J. Am. Pharm. Assoc., Sci. Ed. 40,95-97. GAGE,J. C. (1964).Distribution and excretion of methyl andphenyl mercury salts.Brit. J. Znd. Med. 21,197-202. ISHIKURA,S., and YOKOTA,K. (1963).Analysis of organicmercury. I. Sensitiveanddifferential determination of phenylmercury acetate and inorganic mercury. Chem. Pharm. Bull. (Tokyo)
11,939-942.
MILLER,V. L., KLAVANO,P.A., JESTAD, A. C., andCsoNKA,E. (1961).Absorption, distribution, and excretion of ethylmercuricchloride. Toxicol. Appl. Pharmacol.3,459-468. NISHIKATA,A., TANZAWA,K., TAKEDA,Y., OSAWA,T., and UKITA, T. (1968). Identification and determinationof mercury compoundsin the hair by extraction with acids.EiseiKagaku 14,211-216.
SADAKANE,T. (1964). Studieson the toxicity of mercury. Report 2. Metabolism of ethylmercuricphosphatein animals.SangyoZgaku6,495-502. SLOTTA,K. H., and JACOBI,K. R. (1929).Uber organischeQuecksilbarbasenund ihre Salze. J. Prakt. Chem.120,249-300. SUZUKI,T., MIYAMA, T., andKATSUNUMA, H. (1963).Comparativestudy of bodily distribution of mercury in mice after subcutaneousadministration of methyl, ethyl, and n-propyl mercury acetate.Japan.J. Exptl. Med. 33,277-282. TAKEDA,Y., KUNUGI,T., HOSHINO, O., and UKITA, T. (1968a).Distribution ofinorganic, aryl, and alkyl mercury compoundsin rats. Toxicol. Appl. Pharmacol.13,156-164. TAKEDA,Y., KUNUGI,T., TERAO,T., and UKITA, T. (1968b).Mercury compoundsin the blood of rats treated with ethylmercuric chloride. Toxicol. Appl. Pharmacol.13,165-173. TYNER,E. P., HEIDERBERGER, C., and LEPAGE, G. A. (1953).Intracellular distribution of radioactivity in nucleic acid nucleotidesand proteins following simultaneousadministration of P32and glycine-2-C14.CancerRes.13, 186-203. YEMM, E. W., and COCKING,E. C. (1955). Determination of amino acids with ninhydrin. Analyst 80,209-213.