39,521-529(1977)

I-OXICOLOGYANDAPPLlEDPHARMACOLOGY

VI.

Studies on Selenium-Related Biosynthesis of Dimethyl Selenide Administration of Sodium

Compounds in Rat Liver Selenate’

KATSUHIKONAKAMURO,* YASUYOSHISAYATO,**’ AND *Department of Hygienic Japan, and t Department

Chemistry,

of Environmental Received

YOUKI

after

Oral

0s-f

National Institute of Hygienic Sciences, Setagaya-ku, Tokyo 158, Hygienic Chemistry, Gifu College of Pharmacy, GifL 502, Japan

June 23, 1976;

accepted

October

27,1976

Studieson Selenium-RelatedCompounds.VI. Biosynthesisof Dimethyl Selenidein Rat Liver after Oral Administration of Sodium Selenate. NAKAMURO, K., SAYATO, Y., AND OSE,Y. (1977).Toxicol. Appl. Pharmacol. 39,521-529. Experimentsweremadeto obtain data on the biologicalaction of seleniumin order to establisha standard for water quality for public water supply. Biosynthesisof dimethyl selenidein rat liver after oral administration of Na2Se0.,wasinvestigatedand the volatile seleniumformed was identified. The study showedthat dimethyl selenide,asa respiratory metabolite, wasprobably formed in the rat liver. Differenceswere noted as to dimethyl selenideformation from sodium selenite and sodium selenate in vitro. The test of singleoral administrationof sodiumselenateindicated that dimethyl selenide formation increased progressively up to about 6 mg/kg and then reacheda plateauat this dose.The increasedaccumulation of selenium in the liver after continuous oral administration was found to

stimulatethe methylation of seleniumto dimethyl selenide.When sodium selenate was orally administered to rats, (CH,)$e GLC, and CC-mass spectrometry.

was found

by TLC,

Selenium is an environmental pollutant, and selenium compounds are extensively usedin industry and commerce. The influence of seleniumcompounds on a biological system is therefore important from the standpoint of environmental toxicology. To establish a standard for water quality for public water supply in Japan, we studied the biological actions of selenium, such as acute and subacute toxicity of Na,SeO, in rats (Nakamuro et al., 1974a,b), and absorption, excretion, and distribution of 75Se after a single oral administration of Na, 75Se04to rats (Nakamuro et al., 1972). These studiesshowedthat selenatecausesyellow atrophy ofthe liver in rats. Liver damagefrom industrial seleniumexposure has not been reported, although industrial physicians are alerted to the hepatotoxic effects in animals (Glover et al., 1970). Ganther (1966) has shown that Na, ‘%e03 is converted to (CH,),Se by mouse liver homogenate in vitro, and it is suggestedthat methylation of inorganic selenium 1 Presented, in part, at the 95th Annual Meeting ofthe Pharmaceutical Society of Japan, Nishinomiya, Japan, April 46, 1975. * To whom requests for reprints should be sent. 521 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction Printed in Great Britain

in any form

reserved.

ISSN

0041-008X

522

NAKAMURO,

SAYATO,

AND

OSE

is related to detoxification (McConnell and Portman, 1952a). (CH,),Se is known as a respiratory metabolite (McConnell and Portman, 1952b). However, there is no report concerning the biosynthesis of (CH3)$e in uiuo after the oral administration of Na,SeO,. In the present work, (CH,),Se formation in the rat liver after oral administration of Na,SeO, was examined after a single dose and after continuous administration tests. In addition, for the purpose of confirming (CH,),Se formation in vivo, the volatile selenium from rat liver after oral administration of Na$eO, was isolated and identified by thin-layer chromatography, gas-liquid chromatography, and high-resolution mass spectrometry. METHODS Animals andmaterials. Female Wistar strain rats, weighing 180-220 g, were employed for these experiments. A group of five to six rats was housed in a stainless steel cage. The animals were maintained on a laboratory pellet chow (Oriental Co., Tokyo) for at least 1 week before the experiments. The animals had free access to food and water. Na,SeO, was obtained from Wako Pure Chemical Co., Tokyo. Na,75Se0, and Na275Se0, were purchased from The Radiochemical Centre, United Kingdom. Dimethyl selenide * mercuric chloride [(CH,),Se. HgCl,] was prepared by the method of Bird and Challenger (1942) and recrystallized three times from acetone to yield white crystals, mp, 153°C. Collection of (CH,),Se from rat liver. The object of this experiment was to collect (CH,)$e from the liver of rats given Na, 75Se0, solution (2.5 x lo5 cpm/ml, 0.1 mg of Se/ml). The rats, after a single oral dosage of Na,75 SeO, were sacrificed by exsanguination, their livers were chilled in ice-cold 0.9 % NaCl solution, and a 15 % homogenate was prepared with the same medium in a Potter-Elvehjem homogenizer with a Teflon pestle at 1000 rpm. Loss of (CH&Se during homogenization was not observed. A mixture of 5 ml of this homogenate and 0.5 ml of octanol was placed in a 20-ml glass tube and the tube was bubbled with pure nitrogen gas at 37°C for 1 hr. The flow rate of carrier gas was approximately 200 ml/min. (CH,),Se was trapped in two tubes, each containing 5 ml of saturated mercuric chloride. Radioactivity of this solution was sounted with an Aloka well-type scintillation counter. The data are expressed as percentage of the dose given or as percentage of the dose distributed to the liver. Total 75Se was determined on a separate aliquot of liver homogenate obtained before bubbling with nitrogen. Comparison of (CH&Se formation from Na275Se04 and Na,75Se0,. To compare (CH,),Se formation from Na,SeO, and Na,SeO,, four series of experiments were carried out with the following reaction mixtures: (a) 3 ml of a 15 y0 liver homogenate added with 0.1 ml of Na275Se0, or Na,75Se0, solution (0.5 mg of 75Se/ml, 4 x lo5 cpm/ml); (b) the reaction mixture (a) with the cofactor of methylation reported by Ganther (1966) added ; (c) a 15 % liver homogenate of rats 4 hr after a single oral dose of Na,SeO, or Na,SeO, in 2.1 mg/kg as Se added with 0.1 ml of Na,75Se04 or Na275Se0, solution and incubated under the same condition as (b); (d) the reaction mixture was prepared from rat liver 1 hr after iv administration of 0.5 ml of Na275Se0, or Na275Se0, solution (0.1 mg of 75Se/ml, 2.5 x lo5 cpm/ml). To each of these four mixtures, 0.2 ml of octanol was added as a defoaming agent. (CH&Se was collected by the same

BIOSYNTHESIS

OF DIMETHYL

SELENIDE

523

method as above. These data are expressed as percentage of (CH,),Se formed to the ‘%e added. In vivostudies on biosynthesisof (CHJ,Se. Three series of experiments were performed : time course after single or continuous administration and dose-response relationship of (CH,),Se formation ratio in vivo. The Na, ‘%e04 solution injected was 2.5 x IO5 cpm/O.l mg of 7sSe/ml. The amount of (CH,),Se trapped was determined by counting the radioactivity of its solution with a well-type scintillation counter. The data are expressed as percentage of the dose given or as percentage of the dose distributed to the liver. ZdentiJication of volatile selenium. Fifteen male rats were orally given 5 mg/kg of Na,SeO, for 3 consecutive days and sacrificed by decapitation 4 hr after the final administration. From their livers, a 15% liver homogenate was prepared and volatile selenium was collected in a 0.1% acetone solution of HgCI, as described for the isolation of this metabolite. TABLE I CONDITIONS

Glass column Column temperature Injection temperature Carrier gas Flow rate of carrier gas

FOR GAS-LIQUID

CHROMATOGRAPHY

Porapack-P

5 % DEGS on Shimalite-W

1 m x 3 mm (i.d.) 90°C 185°C N2 100 ml/min

0.5 m x 3 mm (i.d.) 75°C 185°C Nt 30 ml/min

Thin-layer chromatography: Aliquots (30-50 ~1) of the sample were subjected to one-dimensional thin-layer chromatography on a 0.25-mm silica gel plate (5 x 20 cm) activated at 110°C for 60 min. The plate was developed with butyl acetate-acetone (6: 1, v/v). The detecting reagent used for (CH&Se-HgCl, was a 0.005% benzene solution of dithizone. Gas-liquid chromatography: A Model GC-5A gas-liquid chromatograph (Shimadzu Seisakusho, Kyoto) equipped with a flame ionization detector was used. Two glass columns were packed with packing agents of different types of polarity: 5 % DEGS on Shimalite-W (60-80 mesh) and Porapack-P (50-80 mesh). Gas-liquid chromatographic analysis was carried out under the conditions shown in Table 1. An aliquot (10 ~1) of the sample and an acetone solution of authentic (CH,)$e. HgCl, (100 pg/ml) were injected into the gas chromatograph under the above experimental conditions. Volatile selenium was identified by comparing the retention time of its peak with that of the authentic standard. High-resolution mass spectrometry : The high-resolution mass spectrometer (Japan Electron Optics Laboratory, Tokyo) wasconnected with a 20K-type gas chromatograph under the following conditions: 2 m x 3 mm (id.) coiled stainless steel column packed with Porapack-P; column oven temperature, 120°C; ion source, 300°C; electron energy, 75 eV; accelerating voltage, 5 kV. The sample was identified by comparing its spectrum and its fragment ions with those of the standard.

524

NAKAMURO,

SAYATO, AND OSE

RESULTS

Collection of (CH,),Se from Rat Liver Various conditions were examined for efficient and quantitative collection of(CH,),Se in rat liver after oral administration of Na,SeO,. Among the solvents tested, such as ligroine, benzene, saturated mercuric chloride, and water, saturated mercuric chloride

Bubbling

time

(min)

FIG. 1. Relationship between volatile selenium and bubbling time. The rat liver was used 4 hr after administration of Na2?Se04 solution. TABLE COMPARISON OF FORMATION OF (CH&Se

--_. In vitro Na,“SeO (4 Naz7?3e0: Na,“SeO (bf NaZ7’SeO: Naz?SeO (4 Naz75SeOz In vivo (4 -

2 FROM Na2%e04

Doseof pretreatment bx/kg)

AND Na275Se0,

Dimethyl selenide formation Cofactorb (%) ~---

po 2.1 (as Se)Na,SeO, po 2.1 (asSe)Na,Se03

+ + + I

iv 0.05 NaZ7?3e0 4 iv 0.05 NaZ7%e03

-

0 0 1.72 3.30 2.02 13.82 5.84 8.68

n Selenium compounds were added to the rat liver homogenate. b Cofactor which Ganther (1966) used for synthesis of dimethyl selenide by liver fractions. The detailed conditions of (a), (b), (c), and (d) have been described under Methods.

was the most suitable. (CH&Se was volatilized from the rat liver homogenate by bubbling with nitrogen gas. (CH&Se was rapidly releasedwithin 40 min after the start of bubbling and little wasreleasedthereafter (Fig. 1). The optimal bubbling time wasfound to be 60 min, and the collected material did not decomposeor disappear. It was found in in viuo teststhat radioactivity of (CH&Se did not differ when using nitrogen, oxygen, or air. Moreover, (CH,)$e was not isolated when a 15% rat liver homogenate added with Na2’73e0, was incubated by bubbling nitrogen in vitro.

BIOSYNTHESIS

OF DIMETHYL

525

SELENlDE

Comparison of (CH,),Se Formation from Na,75Se0, and Na275Se0, Table 2 shows the data of four series of experiments comparing (CH,),Se formation from Naz7’Se0, and Naz7%e0,. In the in vitro test, Na,75Se0, was found to form (CH,),Se at a higher rate than Na, 75Se04 under conditions (b) and (c) with the cofactor of methylation added. In the in vivo test with rat liver after iv injection of Na,“Se04 or Naz7%e0,, a higher formation of (CH,),Se was also found in rat liver after injection of Na,75Se0,. In Vivo Studies on Biosynthesis of (CH&Se Figure 2 shows the time distribution of (CH,),Se in rat liver after a single oral dose of Na,75Se0,. The radioselenium increased in the liver of both male and female rats and

0

2

4

Time after

administration

6

8

-

(hr)

FIG. 2. Time distribution of (CHJ$e in liver after single oral administration of NazSeO,. O----O, [dimethyl selenide (counts per minute) x lOO/‘%e in liver (counts per minute)] (percentage) (left side); o-o, [dimethyl selenide (counts per minute) x lOO/dose (counts per minute)] (percentage) (left side); x-x, [?5e in liver (counts per minute) x lOO/dose (counts per minute)] (percentage) (right side).

reached a level that was about 20 % of the administered selenium 8 hr after administration. The pattern of (CH,),Se in the male rat liver increased until 2 hr after administration and it reached a plateau thereafter, being about 1.5 % of total 75Se in the liver. (CH,)aSe in the female rat liver also increased, reaching a plateau 30 min after administration, (CH,),Se being0.8 % of total 75Sein the liver. (CH,),Se formation in male and female rats 8 hr after administration was 0.4 and 0.2 %, respectively, of the administered dose. The dose-response relationship of the distribution of (CH,),Se in rat liver after a single oral administration of 0.1-l 1 mg/kg of Na,75Se0, is shown Fig. 3. The distri-

526

NAKAMURO, SAYATO, AND OSE

bution of YSe in the liver showed a rapid decrease with increase in the dose administered. On the other hand, there was a progressive increase in the (CH,),Se formation up to 6 mg/kg of Na,‘%e04, and there was hardly any decrease from these maximum values above this dose level. Figure4 shows the activity of (CH,),Se biosynthesis during continuous oral administration of 5 mg/kg/day of Na,SeO,. When Na,‘?SeO, was administered orally after

- 20

-10

a 0

4

2

6 Dose

8

10

12

h/kg)

3. Dose-response relationship of (CH&Se formation in the liver after single oral administration. w, [dimethyl selenide (counts per minute) x 100 /?Se in liver (counts per minute)] (percentage) (left side); o-o, [dimethyl selenide (counts per minute) x lOO/dose (counts per minute)] (percentage) (left side); x-x, [“Se in liver (counts per minute) x lOO/dose (counts per minute)] (percentage) (right side). FIG.

x

*

*

CJ

0

0 *

Days

6

4

2

of administration

8

of NapSe04

FIG. 4. Effect of (CH&Se formation activity during daily oral administration of 5 mg/kg/day of Na,SeO+ O----O, [dimethyl selenide (counts per minute) x lOO/%e in liver (counts per minute)] (pxcentage) (left side) ; O-O, [dimethyl selenide (counts per minute) x lOO/dose (counts per minute)] (percentage) (left side); x---x, r”Se in liver (counts per minute) x lOO/dose (counts per minute)] (percentage) (right side).

537 1665 1899 4478 1035 1868 4319 1028 1174

79.9364 91.9645 93.9655 94.9747 106.9671 107.9642 109.9613 198.9631 199.9618

mle (observed) 3.5 21.1 21.3 34.7 0.2 0.0 -2.0 -5.1 -6.4

Error (milli-mass

(CH&Se.HgCI,

unit) Ions 710 1938 2449 5445 1311 2437 4879 1052 1404

Intensity (mv)

~----.

79.9411 91.9724 93.9662 94.9788 106.9667 107.9661 109.9594 198.9616 199.9574

mle (observed) 8.2 29.1 22.1 38.8 -0.1 1.9 -3.9 -6.6 -10.9

Error (milli-mass

selenium

unit)

SELENIUM AND

*‘Se, 79.9165 (49.82%);

Volatile

76.9199 (7.58 %); ‘*Se, 77.9173 (23.52%);

Hz7*Se CHs7’Se 13CH378Se CH3*‘Se CzH677Se CZHs7*Se CzHssOSe lQQHg “‘Hg

DNatural distribution of selenium and mercury isotopes: ?e, (16.84%); 200Hg,199.9683 (23.13%).

-.

InteTsity (mv)

3

RESULTS OF GAS CHROMATOGRAPHY-HIGH-RESOLUTION MASS SPECTROMETRY OF VOLATILE SYNTHETIC DIMETHYL SELENIDE*MERCURIC CHLORIDES

TABLE

“‘Hg,

198.9683

H2’%e CH377Se ‘3CH378Se CH3*‘Se C2HG7’Se CzHs7*Se CzHssoSe lQQHg 200Hg

Ions

2 K G 2, a

s 2 x 2 Q Q 2 El ?G 3

528

NAKAMURO,

SAYATO,

AND

OSE

continuous administration, the incorporation of 75Se into the liver showed a rapid increase until 2 days after administration and hardly increased thereafter (about 14 % of the administered dose). After the first dose, all subsequent doses showed about equal but small increments of increased fractional metabolism to (CH,),Se and of fractional increased deposition in the liver. (CH,),Se formation after 8 days was about 8 p/, of total 75Se distributed in the liver and 1% of the administered dose. IdentiJication of Volatile Selenium from Rat Liver When volatile selenium was chromatographed on a silica gel, good resolution was obtained. The metabolite was identified by comparison with the R, value (R, = 0.35) of volatile selenium from the liver and authentic (CH,),Se *HgCl,. The volatile selenium and authentic (CH,),Se *HgCl, were analyzed by gas-liquid chromatography with two different columns. No interfering peaks were encountered in the analysis. The retention time of both materials was the same, 5.8 min on the Porapack-P column and 7.6 min on the 5 % DEGS on Shimalite-W column. Low-resolution mass spectrometry was carried out. Although no parent peak (MC) for (CH,),Se .HgCl, was observed, peaks at m/e 200, 110, 95, and 80 in both materials indicate a possible cleavage. The sample had to be heated to above 300°C before mass peaks were observed, and the selenium-mercury bond may be broken before (CH,),Se. HgCl, becomes volatile enough to be detected. For the identification of fragment ions, high-resolution mass spectrometry was carried out. This analysis indicated (Table 3) that the peak at m/e 200 is mercury and those at m/e 110, 95, and 80 correspond to (CH,),Se, CH,Se, and H,Se, respectively. The mass ratios of the peaks around these masses correspond to the natural distribution of selenium isotopes. More important, the volatile selenium and synthetic (CH,),Se.HgCl, gave approximately the same ratios. From these data, volatile selenium was identified as (CH,),Se. DISCUSSION From our previous reports on the toxic effects of selenate, it is considered that selenium metabolism in the liver holds the key to the toxicity of Na,SeO, in viuo. Much work has been done on the possibility of methylation from selenite in vivo (Ganther, 1966; Palmer et al., 1970; Byard, 1969; McConnell and Portman, 1952b), but the isolation, identification, and formation patterns of (CH&Se from selenate in vivo have not been reported. We therefore examined the mechanism of formation of (CH,),Se in the rat liver after oral administration of Na,SeO,. Our experimental results confirmed that (CH,),Se is not formed in vitro, but is biosynthesized in the rat liver after oral administration of Na,SeO,. Ganther (1966) reported that Na,SeO, was converted to (CH,),Se with a methylation cofactor, but he did not report studies on methylation of Na,SeO, in vivo. Therefore, we compared (CH,),Se formation from Na,SeO, and Na,SeO, in vitro and found that the activity of (CH,),Se formation was obviously higher from Na,SeO, than from Na,SeO, in vitro. Consequently, to study the formation of (CH,),Se from Na,SeO, in viva, the patterns of (CH,),Se formation in rat liver after oral administration of Na,‘%eO, were investigated. The time distribution of (CH,),Se in rat liver after single oral dose of NaZ7%e0,

RIOSYNTIIESISOFDlMETHYL

SELENIDE

529

indicated that the amount of (CH,),Se formed in male rats was twice that in female rats. The ratio of its formation in male and female rats was low, about 0.4 and 0.2x, respectively, as percentage of the administered dose. While our earlier studies showed female rats to be more sensitive to the hepatotoxic effect of selenium than male rats (Nakamuro et al., 1974a), these findings may be closely related to the production of

yellow atrophy of the liver in female rats in the subacute toxicity test. The dose-response tests on the formation of (CH,),Se indicated that the percentage of selenium converted to (CH,),Se increased progressively up to 6 mg/kg of Na,SeO,, with a decreasefrom these maximum values above this dose. These findings may substantiate the hypothesis that appreciable amounts of volatile seleniumare formed only at near toxic levels of selenium (Ganther et al., 1966). The decreaseof (CH,),Se forma-

tion above 6 mg/kg may be due to the appearance of acute toxicity. Basedon the studiesreported here, it is consideredthat methylation of seleniumin the liver is stimulated by the intake of Na,SeO, at or near toxic levels and the increase in the amount of ‘?Se distributed in the liver. Furthermore, it is indicated that (CH,),Se, which was reported as a respiratory product in the exhaled gasesafter an oral dose of selenate (Nakamuro et al., 1972) was already present in the rat liver after oral administration of Na,SeO,. ACKNOWLEDGMENTS The authors thank Dr. Kenzo Kanoda and Mr. HisaoAbe of our laboratory for the mass spectralanalysis. REFERENCES BIRD, M. L., AND CHALLENGER, F. (1942).Potassiumalkaneselenonates and other alkyl derivatives of selenium.J. Chem. Sot. 570-574. BYARD, J. L. (1969). Trimethyl selenide.A urinary metabolite of selenite.Arch. Biochem. Biophys. 130,556-560. GANTHER, H. E. (1966).Enzymic synthesisof dimethyl selenidefrom sodiumselenitein mouse liver extracts. Biochemistry 5, 1089-1098. GANTHER,H.E.,LEVANDER,O. A., AND BAUMANN, C.A.(1966).Dietarycontrolofselenium volatilization in the rat. J. Nutr. 85, 55-60. GLOVER, J. R. (1970).Seleniumandits industrialtoxicology. Indastr. Med. Surg. 39, 50-54. MCCONNELL, K. P., AND PORTMAN, 0. W. (1952a).Toxicity of dimethyl selenidein the rat and mouse.Proc. Sot. Exp. Biol. Med. 79,230-231. MCCONNELL, K. P., AND PORTMAN, 0. W. (1952b).Excretion of dimethyl selenideby the rat. J.Biol. Chem. 195,277-282. NAKAMURO,K., SAYATO,Y.,TONOMURA, M., AND OSE, Y.(1972).Studies onseleniumrelated compounds.II. The metabolicfate of selenate(Na, ‘?SeO& Eisei Kagaku 18, 368-373. NAKAMURO, K., SAYATO, Y., TONOMURA, M., AND OSE,Y. (1974a).Studieson seleniumrelated compounds.III. Acute and subacutetoxicity of sodiumselenate.Eisei Kagaku 20, 29-35. NAKAMURO,K.,SAYATO,Y.,TONOMURA, M., AND OSE, Y.(1974b).Studieson seleniumrelated compounds.IV. Acute and subacutetoxicity of sodiumselenate.Eisei Kagaku 20, 75-80. PALMER, L. S., GUNSALUS, R. P., HALVERSON, A. W., AND OLSON, 0. E.(1970). Trimethylselenoniumion asa generalexcretory product from seleniummetabolismin the rat. Biochim. Biophys. Acta 208,260-266.

Studies on selenium-related compounds. VI. Biosynthesis of dimethyl selenide in rat liver after oral administration of sodium selenate.

39,521-529(1977) I-OXICOLOGYANDAPPLlEDPHARMACOLOGY VI. Studies on Selenium-Related Biosynthesis of Dimethyl Selenide Administration of Sodium Comp...
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