Nucl. Med. Biol. Vol. 19, No. 4, pp. 423429, Inr. .I. Radial. Appl. Instrum. Part B
1992 Copyright
0
0883-2897/92 S5.00 + 0.00 1992 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
Time-dependent Distribution of Selenate and Selenite Selenium in Male C57L/J Mice MUHAMMAD Department
ASHRAF TARIQ* and IVOR L. PREISSt
of Chemistry,
Rensselaer
Polytechnic
Institute,
Troy,
NY 12180, U.S.A.
(Received 4 November 1991) The distribution and retention of selenate and selenite in C57L/J mice was investigated using 75Se radioisotope. The comparison is made with the normal distribution of the element determined by using radioisotope-induced x-ray fluorescence, RIXRF methods. The distribution of the two oxidation states measured as activity of “Se was almost identical but differed from the normal trace elemental profile, TEP. The excretion rates of the two selenium species in the initial phase are different with more selenate being excreted than selenite in the first 2 days. It was found that the wholebody excretion rates followed a pure first-order pattern this was not true for the individual organs of the animals.
Introduction
(Behne and Wolters, 1983; Hopkins et al., 1966; Kinder et al., 1988; McConnell, 1941; Oster et al., 1988; Thorlacius-Ussing and Jensen, 1988). The information on the comparison of the distribution and excretion of selenite and selenate is limited. Despite a similar bioavailability exhibited by the selenate and selenite, the two chemical forms differ in their mode of absorption and transport (Beilstein and Whanger, 1988; Korpela, 1988; Wolfram et al., 1986, 1988). It has been observed that similar doses of selenite and selenate have different effects on the growth of rats (Schroeder, 1967), but, no significant difference in selenium blood levels has been observed in humans given dietary selenite and selenate over a period of 3 months (Clausen and Nielsen, 1988). The observations reported to date have involved a single organ or have been studied in vitro. More insight into the subject can be obtained in a comprehensive study in which various organs can be examined simultaneously. In the present study, the distribution of injected 75Se as selenate and selenite, in various organs of C57L/J mouse has been investigated. A comparison is made with content of selenium in various tissues resulting from normal modes of ingestion and metabolism. These concentrations have been determined using a radioisotope-induced x-ray fluorescence method (Tariq and Preiss, 1991). The administered selenium levels were adjusted to be such that the normal distribution of selenium ingested through normal dietary modes will not be perturbed. Thus, perturbations due only to the oxidation state of selenium can be deduced.
It has been reported that selenite and selenate are not naturally-occurring forms of selenium in foods but are common forms found in water. These oxidation states are readily metabolized to forms with high biological activity such as, selenocysteine (Ganther, 1986). Concern about the presence of selenium in its various forms has grown with evidence of their high concentrations in waters of Kesterson Refuge, Calif. Higher mortality and deformity rates observed in aquatic life and water fowl were attributed to the high concentrations of selenium in the refuge water (Deveral and Millard, 1988; Robberecht and Grieken, 1982; Tanji et al., 1986). 94% of the total inorganic selenium in these waters is reported to be in the form of selenate, an oxidation state of 6 for the element. The oxidation state of 4, i.e. selenite, is considered to be more biologically active (Schroeder, 1967). It has been suggested that selenate enters the human and animals through a selenite pathway (Ganther, 1986). Clear evidence of this is still to be observed, however. The problem demands a thorough investigation of the physiological and metabolic differences between the two inorganic forms of selenium. The distribution of the inorganic and organic chemical forms of selenium have been found to be different as reported in human and animal studies
*On leave from Institute of Chemistry, Punjab, Lahore, Pakistan. TAuthor for correspondence.
University
of the
423
MUHAMMAD ASHRAF TARIQ and
424
Materials [“Se]Selenate
and Methods
IVORL. Pasiss
Table I. Selenium content in blood and some organ samples of a C57L/J mouse
and [75Se]selenite solutions
0.203 mCi of [75Se]selenous acid (DuPont)
was refluxed with 3 mL of 2 M HCI for 5 h and the solution was evaporated to dryness. After the addition of 3 mL of deionized water the evaporation was repeated. 1 mL of water was then added and radiochemical purity was checked by following the TLC procedure described by Ganther (1968). The purity of selenite was found to be more than 98%. 5 mL of phosphate buffer saline (PBS) containing 1Opg of carrier sodium selenite (Aldrich) were added and the pH of solution was adjusted to 7.4 with NaOH or HCI. The volume was made to 10 mL with PBS of pH = 7.4. Each mouse was injected with 1OOpL of this solution giving a total dose of 0.1 pg selenite with an activity of 2pCi. Another 0.202mCi portion of selenous acid was refluxed with an oxidation mixture containing 3 mL of concentrated HNOs and 1 mL of 70% perchloric acid for 5 h. The procedure was repeated after evaporation to near dryness. The radiochemical purity of selenate was checked by the above mentioned procedure and was found to be more than 96%. Steps in the production of selenate solution were identical except for the substitution of Na,SO, for Na,SO,. Procedure 53 male C57L/J mice (Jackson Laboratories, Anhorbour, Main, U.S.A.) (Heinenger and Dorey, 1980) with body masses of 22 + 2 g were kept under standard laboratory conditions in an animal room. Roden blax pellet food containing 0.1 pg/g dry mass selenium (determined by radioisotope x-ray fluorescence) and tap water were available at all times. After 4 weeks 5 mice were sacrificed and mean selenium content in various organs was determined and radioisotopeemploying preconcentration, induced x-ray fluorescence techniques according to the procedure described elsewhere (Tariq and Preiss, 1992). The remaining 48 mice (weighing 25 + 2 g) were equally divided into group A and group B for the time-dependent distribution study of radioactive selenite and selenate selenium, respectively. The group A mice were injected interaocularly with 100 PL (2 PCi) of sodium selenite solution and group B mice with 100 PL (2 PCi) of sodium selenate solution. The low level doses (c. 4 ppb) of selenium were employed so as not to disturb the normal physiological level of the element. The interaocular injection resulted in rapid distribution throughout the whole body of the animal. The solutions were injected under membutal anesthesia on the day designated as day “zero” of the experimental period. The previously described diet was continued after random division of mice into further subgroups of 3 mice each. Whole body counting of radioactive selenium for each mouse was performed 1 h after injection. A Harshaw 7.63 x 7.63 cm
Se content @g/g f SD*) Tissue Liver Kidneys Spleen Testicles Digestive system Brain Pancreas Lungs Heart Muscle Whole blood Skin
Wet mass
Dry mass
0.7OkO.12 1.33- * 0.21 0.35 0.21 0.17 f 0.05 0.11 f 0.02 0.31 * 0.08 0.29 i 0.1 I 0.23 0.12+0.04 0.37 * 0.10 0.15 k 0.06
2.42 f 0.50 5.54 + 1.05 1.40 0.80 0.71 * 0.20 I .45 1.16 0.82 0.57 * 0.15
*SD = standard deviation. The organs have been pooled where SD is not reported.
NaI(T1) crystal was used to obtain whole body counts. Anesthetized mice were placed in a constraining rectangular container with the crystal along the upper face. This insured reproducible geometry. The mice were sacrificed after 1, 2 h and 1, 2, 4, 8, 16 and 32 days, one subgroup at a time. Each mouse was counted once again (except subgroup 1) before sacrifice to determine total body retention after the respective period. The blood and portions of organ tissue (listed in Table 1) were collected in preweighed cylindrical acrylic vials and capped. The vials were counted in a snug fitting 1.7 cm diameter well that had been cut into another 7.63 x 7.63 cm Harshaw NaI(TI) crystal. The whole body detector and the cylindrical tissue detector each had its own preamplifier and spectroscopy amplifier. Both were coupled through a Canberra Industries Model 8075 ADC to an IBM 9000 computer system, used for data acquisition and analysis. Intensity was determined by integrating between 10th maximum values for 265 and 280 keV y-energy peaks for 75Se. Background was subtracted using a linear integration method (Preiss and Keenan, 1988). The efficiencies of whole body and well detectors were calibrated and matched in the calculations. The weights of blood and tissues counted were kept within 0.2 g of each other in order to minimize absorption differences within the sample. Results Distribution of selenium in the tissues
The mean selenium content in the blood and various tissues of 5 normal C57L/J mice under the described dietary conditions is given in Table 1. Table 2 shows the mean weights of tissues, percent distribution of selenium and percent distribution of radioactive selenite and selenate 1 day after the injection. The masses of blood and various tissues were assessed in order to calculate the percent distribution of selenium normally present and that found following injection of radioactive selenium. In some cases only a portion of tissue mass was analyzed or counted for 75Se activity. The digestive system includes stomach,
Distribution
of selenate
Table 2. Percent content
and sclenite selenium
in mice
of Se, (75Se]selenite and [“Se]selenate
425
in mouse tissues % Se content*
Tissue
Wet weight (g)
Liver Kidney Spleen Testicles Gastrointestinal Brain Pancreas Lung Heart Muscle Whole blood
1.465+0.115 0.535 * 0.053 0.095 f 0.018 0.145 f 0.021 2.800 + 0.931 0.384 f 0.027 0.225 k 0.050 0.285 + 0.075 0. I61 + 0.026 11.250 * 0.900 2.451 * 0.238
tract
Selenium
[“Se]selenite
[“Se]selenate
21.60 14.89 0.69 0.63 9.98 0.88 I .47 1.74 0.78 28.3 I 19.02
28.87 20.19 0.17 0.96 12.31 0.27 0.99 3.11 0.87 13.50 18.83
30.82 15.76 0.73 0.90 15.65 0.29 1.16 1.94 0.92 13.56 18.26
*For the Se content in normal mouse, values are the mean of 5 mice while it is the average of 3 in others. ‘5Se-percentage contents are for 24 h after interaocular injections. Errors for all Se contents were within 10% of each other. For an explanation please see the text.
colon and small intestine. Muscle was assessed on 45% of the body weight bases as was assessed for rats by others (Behne and Walters, 1983). The radioactivity in the tissue was normalized to the whole body activity measured 1 h after injection
and incorporated proper efficiency corrections for the detectors. The term “specific activity” used in the text indicates activity per minute per gram of the tissue divided by the wholebody activity per minute per gram of body weight.
Time dependent distribution of Se(IV) in normal mice LEGEND
0.04
0.68
1.60
2.60 4.00 Time (day)
8.60
18.0
3i.0
’
LEGEND rZa Stomach m Brain 5I
0.04
0.08
1.00
2.00 4.00 Time (day)
8.00
18.0
Fig. 1. (a, b) The average content of [75Se]selenite for 3 mice in a particular injection.
Specific activity
= cpm/g
(organ)/cpm/g
wholebody
Muscle
31.0
organ at different 1 h after injection.
times after
MUHAMMAD ASHRAFTARIQand IVORL. Pasiss
426
The specific activity of [75Se]selenite and [75Se]selenate selenium as a function of time in some of the individual organs is shown in Figs l(a, b) and 2(a, b),
respectively. Liver contains maximum activity at earlier periods in the experiment and was ten times as large as whole body activity for both the selenite and the selenate. The kidney content begins to exceed that of liver 24 h post-injection but continues to be less than the initial kidney content. The specific activities in the kidney after 24 h were 3.79 and 2.68 times the wholebody activity for selenite and selenate selenium, respectively. The lowest levels of activity were found in brain and muscle over the whole period of investigation. Blood, spleen, lungs and heart contents differ among each other by small factors and were random around the wholebody activity. The content of two selenium species in testicles was low in the beginning but became significant after a few days. Testicles accumulated higher concentrations of selenate than of selenite. The specific activity in kidney, spleen, blood and lungs were higher for selenite than for selenate after
24 h. The digestive system retained relatively more selenate than selenite. At the end of the study period, all tissues analyzed, except testes and brain, have higher selenite content as compared to selenate. Retention of selenium The retention of [” Selselenite and [” Selselenate after a single interaocular injection was monitored in blood and 10 tissue samples from animals sacrificed at fixed time intervals over a period of 31 days. The results are shown in Figs 3 and 4(a-d) for a few of the tissues investigated. Wholebody A rapid excretion of both selenite and selenate is observed in the first 24 h. The differences in the wholebody retention behavior become significant after 2 days with selenite content higher than the selenate. Nonetheless, Se(IV) and Se(M) excretion follow a similar pattern of rapid excretion followed by a slower phase. The excretion in the latter phase follows a first-order kinetic behavior with biological
Time dependent distribution of Se(V1) in normal mice LEGEND eZa Liver m Kidney U Whole Blood C!Z¶ Spleen C?Sl Testicles
Time (day) LEGEND tZZl Stomach
0.04
0.08
1.00
2.00 4.00 Time (day)
8.00
18.0
31.0
Fig 2. (a, b) The average content of [75Selselenate for 3 mice in a particular organ at different times after injection. Specific activity = cpm/g (organ&pm/g wholebody 1 h after injection.
Distribution of selenate and selenite selenium in mice
427 1: l -Liver-Se IV I 2: o-Liver-Se &A
1: .-Whole body,Se 2: o -Whole body,Se
3 -1.0
I
I
12.2
5.6
I
I
I
18.8
25.4
32.0
2)
-1.0
1
I
I
5.5
120
18.5
25.0
31.5
55
12.0
18 5
25.0
31.5
65
li.0
lb.5
2b.o
3i 5
Days Post Injection Fig. 3. Wholebody retention of selenite and selenate at different times after injection. Normalized to initial wholebody activity. Each point represents the mean body retention of 3 mice at the time of sacrifice. The individual data points were within 10%.
half-lives of 17 days for the two selenium species (Fig. 3). Liver
The excretion rate of the two selenium species in liver is very rapid in the initial phase where more than 80% activity is excreted within 1 day [Fig. 4(a)]. A slow change is observed in the next 15 days which follows another rapid excretion. After 30 days only 3-S% of initial dose is retained. The excretion does not follow the first-order kinetics and the concentration decreased to half the initial amount in approx. 16 days in the slower phase of the excretion.
3
-10
r
Kidney and stomach
The kidney retention was more uniform as compared to the liver throughout the experimental period with a lower content for selenate up to the end of the study [Fig. 4(b)]. In the second phase the concentration decreased by a factor of 2 in 11 and 12 days for selenite and selenate, respectively. The distribution of selenate and selenite in the gastrointestinal tract (stomach and the intestines only) exhibited a complementary characteristic for kidney. The selenite content in the stomach was lower than selenate, the case in kidney is opposite. Yet, the excretion half-lives of the stomach were around 9 days for both species.
1 _I
-1.0
Lungs, spleen and wholeblood
The retention in the lungs, spleen and wholeblood fluctuated and did not follow any specific pattern [Fig. 4(c)]. No rapid depletion phase was observed in these organs and the observation was common for both selenium species.
Jr-\ IS
Testicles
Rather than a depletion, an accumulation was observed in this organ for both chemical forms of selenium. After 30 days, 50% of selenite and 70% of initial selenate were still retained [Fig. 4(d)].
LO.0
15.5
21.0
20.5
Days Post Injection Fig. 4. (ad) Retention of selenite and selenate at different times after injection. Normalized to initial wholebody activity. Each point is the mean organ retention of 3 mice. The individual data points were within 10%.
MUHAMMADASHRAF TARIQand
428 Discussion
The general character of the distribution of intrinsic selenium in various tissues of C57L/J mouse (Table 1) is similar to the pattern observed in other studies reported in the literature (Behne and Wolters, 1983; Kinder et al., 1988; McConnell, 1941). The percent distribution, however, is different as shown in Table 2. This can, in part, be accounted for because the gastrointestinal tract and contents were also included as one of the organs in this study. This inclusion was necessary since the mechanism for quantitative absorption and simulation of the IV and VI oxidation states via the digestive tract are different (Korpela, 1988; Wolfram et al., 1986, 1988). The total content of selenium was highest in muscle, followed, in order, by liver, blood, kidney and gastrointestinal tract and its contents. Selenium content in the mouse kidney and testes differ, from that which has been reported for rats (Behne and Wolters, 1983; Hopkins et al., 1966). The distribution of [75Se]selenite or [“Selselenate 1 day after it is administered by injection does not follow the pattern of intrinsic selenium. The content in the blood is similar but the muscle pool contains one half when normal ingestion is compared. The liver, kidney and digestive system contents are higher, exhibiting the preferential retention of the selenium supplemented as oxyanions by injection. Although the initial specific activity was similar, the content of [“Se]selenite in the tissues analyzed was found to be higher than [75Selselenate throughout the experimental period [see Figs l(a, b) and 2(a, b) for comparison]. The distribution patterns of selenite and selenate are amazingly similar except for differences in excretory organs. It is the absolute concentration in a given tissue type that differs. The percentage of selenite retained by the kidney was always higher than that of selenate. The opposite was observed in the stomach. The specific activity of urine and feces was monitored around 24 h after injection. The activity of selenite was higher in urine and that of selenate in the feces. (Total content after an extended period is not available in this experiment.) It appears that more selenite is excreted through the kidney, while more selenate is retained by the stomach and the digestive system in the initial 24 h phase. The wholebody and organs follow an excretion pattern that exhibits two distinct rate dependencies for both of the radioactive selenium species. A rapid initial phase within the first 24 h and a slower phase after that [Figs 3 and 4(a-d)]. Similar behavior has been reported in other studies (Thorlacius-Ussing and Jensen, 1988). The excretion rate for organs in the slower phase may be first order if an uncertainty of f20% were included for each data point. However, the uncertainty among individual mice does not exceed + 10%. It therefore appears that the mechanism
IVORL. Pasrss
of organ excretion in the slower phase may be complex. The fluctuations in the elimination curves for various organs can be explained if selenium, in one chemical form, is deposited in the tissue while in another form it is released at a different rate. This is evident from the fact that wholebody excretion follows a first-order kinetic pattern for selenite and selenate (Fig. 3). Wholebody selenate retention was 13% higher than selenate 1 day after injection. After that the excretion of selenate was faster and the wholebody selenite specific activity was about 13% higher than that of the selenate after 8 days. However, the slopes of the two curves are similar (the rates are similar but the concentrations differ). The relative accumulation in testes was much higher in our experiment than reported for rats by others. This difference may arise from the very low dose of selenium administered in this study in an attempt to keep the physiology of the mouse unperturbed by not raising the concentration of the toxin (i.e. selenium). It is well known now that the distribution of selenium is dose-dependent (Hopkins et al., 1966). Two species seem to differ only in the initial phase. The higher retention of whole body selenate in the initial phase can be explained if the contents of the gastrointestinal tract remain within the mouse body for a longer period than urine. The percent distribution of radioactive selenate or selenite in the liver after 1 day is almost the same suggesting a common chemical form at this site of high metabolic activity for selenium. This in turn suggests that selenate is converted to selenite before being incorporated in protein. Because the content of selenite is always higher [Fig. 4(a)] it could be inferred that selenite has some direct pathway while selenate must be converted into selenite within the initial time phase following injection. It is also clear that not all of the selenate follows the selenite pathway. More detailed studies in the very early stage and sensitive in ciuo or in vitro speciation techniques are required to quantify these differences. Conclusions The specific metabolic chemistry of selenate differs from that of selenite. The apparent reduction to the plus IV state in the liver is but one indication of this difference in pathway. It is possible that many of the contradictory conclusions that appear in the literature have their basis in the fundamental chemical differences of the two oxyanions. It must be expected that SeO:- would follow the chemistry of sulfur (SO:-) while the thermodynamics of the oxidation reduction complex for SeO:- are significantly different than those of sulfite. Thus, oxidation state IV must follow a pathway independent of sulfur, and independent of the carrier effects of the sulfur oxyanions.
Distribution of selenate and selenite selenium in mice Acknowledgements-We
want to acknowledge the support from USPHS Grant 7RR0 7104. M. A. Taria wishes to thank the Government of Pakistan for its continuing support. Also support from Zeta International Ltd is gratefully acknowledged.
429
the female ICR mouse. Bull. envir. Contam. Toxicol. 40, 425.
Korpela H. (1988) Comparative effect of selenate and selenite on serum selenium concentration and glutathione peroxidase activity in selenium-depleted rats. Ann. Nutr. Metab. 32, 347.
References Arenholt-Bindslev D., Larsen B., Abdulla M. and Jepsen A. (1988)Antagonistic effect of selenite on tumor promotor induced cell proliferation in cultures of rat tongue eoithelium. Biol. Trace Elem. Res. 15. 2.57. Behne D. and Wolters W. (1983) Distribution of selenium and gluta thione peroxidase in the rat. J. Nutr. 113,456. Beilstein M. A. and Whanner P. D. (1988) Glutathione peroxidase activity and chemical forms of selenium in tissue of rats given selenite or selenomethionine. J. Inorg. Biochem. 33, 3 1. Clausen J. and Nielsen S. A. (1988) Comparison of whole blood selenium values and erythrocyte glutathione peroxidase activities of normal individuals on supplementation with selenite, selenate, L-selenomethionine and high selenium yeast. Biol. Trace Elem. Res. 15, 125. Deverel S. J. and Millard S. P. (1988) Distribution and mobility of selenium and other trace elements in shallow ground water of the western San Joaquin Valley, _ California. Envir. Sci. Technol. 22, 697. _ Ganther H. E. (1968) Selenotrisulfides. Formation bv the reaction of thibls with selenous acid. Biochemistry 7, i898. Ganther H. E. (1986) Pathways of selenium metabolism including respiratory excretory products. J. Am. CON. Toxicol. 5,
1.
Heinenger H. J. and Dorey J. J. (1980) Handbook on Genetically Standardized Jax Mice. The Jackson Laboratory, Bar Harbour, Mass. Hopkins L. L., Pope A. L. and Baumann C. A. (1966) Distribution of microgram quantities of selenium in the tissues of rat, and effect of previous selenium intake. J. Nutr. 88, 61.
Ihnat M. (1989) Occurrence and Distribution of Selenium, p. 171. CRC Press, Boca Raton, Fla. Kinder S. D., Corey N. C., Razniak S. L. and Hogan G. R. (1988) Time-dependent distribution of sodium selenite in
McConnell K. P. (1941) Distribution and excretion studies in the rat after a single subtoxic subcutaneous injection of sodium selenate containing radioselenium. J. Biol. Chem. 141, 427. Oster O., Schmiedel G. and Prellwitz W. (1988) The organ distribution of selenium in German adults. Biol. Trace Elem. Res. IS, 23.
Preiss I. L. and Keenan J. F. (1988) Time dependent zinc distribution in the development of the BW7756 murine hepatoma. Nucl. Med. Biol. 15, 361. Robberecht H. and Grieken R. V. (1982) Selenium in environmental waters: determination, speciation and concentration levels. Talanta 29, 823. Schoental R. (1968) 7sSe in the harderian glands and brown fat of rats given sodium selenite labeled with 7SSe.Nature Zig, 294. Schroeder H. A. (1967) Effect of selenate, selenite and tellurite on the growth and early survival of mice and rats. J. Nutr. 92, 334.
Tanji K., Lauchli A. and Meyer J. (1986) Selenium in the San Joaquin Valley. Environment 28, 6. Tariq M. A. and Preiss I. L. (1991) Growth inhibition of Bw7756 murine hepatoma by selenium. Cancer J. 4, 49. Tariq M. A. and Preiss I. L. (1992) Determination of selenium in tissue of C57L/J mouse using radioisotope induced X-ray fluorescence. To appear. Thorlacius-Ussing 0. and Jensen F. T. (1988) Selenium in the anterior pituitary of the rat after a single injection of “Se-sodium selenite. Biol. Trace Elem. Res. 15, 277. Wolfram S., Grenacher B. and Scharrer E. (1988) Transport of selenate and sulfate across the intestinal brush-border membrane of pig jejunum by two common mechanisms. Q. J. Exp. Physiol. 73, 103. Wolfram S., Arduser F., Scharrere E. and Schneider B. (1986) Transport of selenate and selenite across brushborder membrane of rat and sheep small intestine. Biol. Trace Elem. Res. 9, 281.
1992 Copyright
0
0883-2897/92 S5.00 + 0.00 1992 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
Time-dependent Distribution of Selenate and Selenite Selenium in Male C57L/J Mice MUHAMMAD Department
ASHRAF TARIQ* and IVOR L. PREISSt
of Chemistry,
Rensselaer
Polytechnic
Institute,
Troy,
NY 12180, U.S.A.
(Received 4 November 1991) The distribution and retention of selenate and selenite in C57L/J mice was investigated using 75Se radioisotope. The comparison is made with the normal distribution of the element determined by using radioisotope-induced x-ray fluorescence, RIXRF methods. The distribution of the two oxidation states measured as activity of “Se was almost identical but differed from the normal trace elemental profile, TEP. The excretion rates of the two selenium species in the initial phase are different with more selenate being excreted than selenite in the first 2 days. It was found that the wholebody excretion rates followed a pure first-order pattern this was not true for the individual organs of the animals.
Introduction
(Behne and Wolters, 1983; Hopkins et al., 1966; Kinder et al., 1988; McConnell, 1941; Oster et al., 1988; Thorlacius-Ussing and Jensen, 1988). The information on the comparison of the distribution and excretion of selenite and selenate is limited. Despite a similar bioavailability exhibited by the selenate and selenite, the two chemical forms differ in their mode of absorption and transport (Beilstein and Whanger, 1988; Korpela, 1988; Wolfram et al., 1986, 1988). It has been observed that similar doses of selenite and selenate have different effects on the growth of rats (Schroeder, 1967), but, no significant difference in selenium blood levels has been observed in humans given dietary selenite and selenate over a period of 3 months (Clausen and Nielsen, 1988). The observations reported to date have involved a single organ or have been studied in vitro. More insight into the subject can be obtained in a comprehensive study in which various organs can be examined simultaneously. In the present study, the distribution of injected 75Se as selenate and selenite, in various organs of C57L/J mouse has been investigated. A comparison is made with content of selenium in various tissues resulting from normal modes of ingestion and metabolism. These concentrations have been determined using a radioisotope-induced x-ray fluorescence method (Tariq and Preiss, 1991). The administered selenium levels were adjusted to be such that the normal distribution of selenium ingested through normal dietary modes will not be perturbed. Thus, perturbations due only to the oxidation state of selenium can be deduced.
It has been reported that selenite and selenate are not naturally-occurring forms of selenium in foods but are common forms found in water. These oxidation states are readily metabolized to forms with high biological activity such as, selenocysteine (Ganther, 1986). Concern about the presence of selenium in its various forms has grown with evidence of their high concentrations in waters of Kesterson Refuge, Calif. Higher mortality and deformity rates observed in aquatic life and water fowl were attributed to the high concentrations of selenium in the refuge water (Deveral and Millard, 1988; Robberecht and Grieken, 1982; Tanji et al., 1986). 94% of the total inorganic selenium in these waters is reported to be in the form of selenate, an oxidation state of 6 for the element. The oxidation state of 4, i.e. selenite, is considered to be more biologically active (Schroeder, 1967). It has been suggested that selenate enters the human and animals through a selenite pathway (Ganther, 1986). Clear evidence of this is still to be observed, however. The problem demands a thorough investigation of the physiological and metabolic differences between the two inorganic forms of selenium. The distribution of the inorganic and organic chemical forms of selenium have been found to be different as reported in human and animal studies
*On leave from Institute of Chemistry, Punjab, Lahore, Pakistan. TAuthor for correspondence.
University
of the
423
MUHAMMAD ASHRAF TARIQ and
424
Materials [“Se]Selenate
and Methods
IVORL. Pasiss
Table I. Selenium content in blood and some organ samples of a C57L/J mouse
and [75Se]selenite solutions
0.203 mCi of [75Se]selenous acid (DuPont)
was refluxed with 3 mL of 2 M HCI for 5 h and the solution was evaporated to dryness. After the addition of 3 mL of deionized water the evaporation was repeated. 1 mL of water was then added and radiochemical purity was checked by following the TLC procedure described by Ganther (1968). The purity of selenite was found to be more than 98%. 5 mL of phosphate buffer saline (PBS) containing 1Opg of carrier sodium selenite (Aldrich) were added and the pH of solution was adjusted to 7.4 with NaOH or HCI. The volume was made to 10 mL with PBS of pH = 7.4. Each mouse was injected with 1OOpL of this solution giving a total dose of 0.1 pg selenite with an activity of 2pCi. Another 0.202mCi portion of selenous acid was refluxed with an oxidation mixture containing 3 mL of concentrated HNOs and 1 mL of 70% perchloric acid for 5 h. The procedure was repeated after evaporation to near dryness. The radiochemical purity of selenate was checked by the above mentioned procedure and was found to be more than 96%. Steps in the production of selenate solution were identical except for the substitution of Na,SO, for Na,SO,. Procedure 53 male C57L/J mice (Jackson Laboratories, Anhorbour, Main, U.S.A.) (Heinenger and Dorey, 1980) with body masses of 22 + 2 g were kept under standard laboratory conditions in an animal room. Roden blax pellet food containing 0.1 pg/g dry mass selenium (determined by radioisotope x-ray fluorescence) and tap water were available at all times. After 4 weeks 5 mice were sacrificed and mean selenium content in various organs was determined and radioisotopeemploying preconcentration, induced x-ray fluorescence techniques according to the procedure described elsewhere (Tariq and Preiss, 1992). The remaining 48 mice (weighing 25 + 2 g) were equally divided into group A and group B for the time-dependent distribution study of radioactive selenite and selenate selenium, respectively. The group A mice were injected interaocularly with 100 PL (2 PCi) of sodium selenite solution and group B mice with 100 PL (2 PCi) of sodium selenate solution. The low level doses (c. 4 ppb) of selenium were employed so as not to disturb the normal physiological level of the element. The interaocular injection resulted in rapid distribution throughout the whole body of the animal. The solutions were injected under membutal anesthesia on the day designated as day “zero” of the experimental period. The previously described diet was continued after random division of mice into further subgroups of 3 mice each. Whole body counting of radioactive selenium for each mouse was performed 1 h after injection. A Harshaw 7.63 x 7.63 cm
Se content @g/g f SD*) Tissue Liver Kidneys Spleen Testicles Digestive system Brain Pancreas Lungs Heart Muscle Whole blood Skin
Wet mass
Dry mass
0.7OkO.12 1.33- * 0.21 0.35 0.21 0.17 f 0.05 0.11 f 0.02 0.31 * 0.08 0.29 i 0.1 I 0.23 0.12+0.04 0.37 * 0.10 0.15 k 0.06
2.42 f 0.50 5.54 + 1.05 1.40 0.80 0.71 * 0.20 I .45 1.16 0.82 0.57 * 0.15
*SD = standard deviation. The organs have been pooled where SD is not reported.
NaI(T1) crystal was used to obtain whole body counts. Anesthetized mice were placed in a constraining rectangular container with the crystal along the upper face. This insured reproducible geometry. The mice were sacrificed after 1, 2 h and 1, 2, 4, 8, 16 and 32 days, one subgroup at a time. Each mouse was counted once again (except subgroup 1) before sacrifice to determine total body retention after the respective period. The blood and portions of organ tissue (listed in Table 1) were collected in preweighed cylindrical acrylic vials and capped. The vials were counted in a snug fitting 1.7 cm diameter well that had been cut into another 7.63 x 7.63 cm Harshaw NaI(TI) crystal. The whole body detector and the cylindrical tissue detector each had its own preamplifier and spectroscopy amplifier. Both were coupled through a Canberra Industries Model 8075 ADC to an IBM 9000 computer system, used for data acquisition and analysis. Intensity was determined by integrating between 10th maximum values for 265 and 280 keV y-energy peaks for 75Se. Background was subtracted using a linear integration method (Preiss and Keenan, 1988). The efficiencies of whole body and well detectors were calibrated and matched in the calculations. The weights of blood and tissues counted were kept within 0.2 g of each other in order to minimize absorption differences within the sample. Results Distribution of selenium in the tissues
The mean selenium content in the blood and various tissues of 5 normal C57L/J mice under the described dietary conditions is given in Table 1. Table 2 shows the mean weights of tissues, percent distribution of selenium and percent distribution of radioactive selenite and selenate 1 day after the injection. The masses of blood and various tissues were assessed in order to calculate the percent distribution of selenium normally present and that found following injection of radioactive selenium. In some cases only a portion of tissue mass was analyzed or counted for 75Se activity. The digestive system includes stomach,
Distribution
of selenate
Table 2. Percent content
and sclenite selenium
in mice
of Se, (75Se]selenite and [“Se]selenate
425
in mouse tissues % Se content*
Tissue
Wet weight (g)
Liver Kidney Spleen Testicles Gastrointestinal Brain Pancreas Lung Heart Muscle Whole blood
1.465+0.115 0.535 * 0.053 0.095 f 0.018 0.145 f 0.021 2.800 + 0.931 0.384 f 0.027 0.225 k 0.050 0.285 + 0.075 0. I61 + 0.026 11.250 * 0.900 2.451 * 0.238
tract
Selenium
[“Se]selenite
[“Se]selenate
21.60 14.89 0.69 0.63 9.98 0.88 I .47 1.74 0.78 28.3 I 19.02
28.87 20.19 0.17 0.96 12.31 0.27 0.99 3.11 0.87 13.50 18.83
30.82 15.76 0.73 0.90 15.65 0.29 1.16 1.94 0.92 13.56 18.26
*For the Se content in normal mouse, values are the mean of 5 mice while it is the average of 3 in others. ‘5Se-percentage contents are for 24 h after interaocular injections. Errors for all Se contents were within 10% of each other. For an explanation please see the text.
colon and small intestine. Muscle was assessed on 45% of the body weight bases as was assessed for rats by others (Behne and Walters, 1983). The radioactivity in the tissue was normalized to the whole body activity measured 1 h after injection
and incorporated proper efficiency corrections for the detectors. The term “specific activity” used in the text indicates activity per minute per gram of the tissue divided by the wholebody activity per minute per gram of body weight.
Time dependent distribution of Se(IV) in normal mice LEGEND
0.04
0.68
1.60
2.60 4.00 Time (day)
8.60
18.0
3i.0
’
LEGEND rZa Stomach m Brain 5I
0.04
0.08
1.00
2.00 4.00 Time (day)
8.00
18.0
Fig. 1. (a, b) The average content of [75Se]selenite for 3 mice in a particular injection.
Specific activity
= cpm/g
(organ)/cpm/g
wholebody
Muscle
31.0
organ at different 1 h after injection.
times after
MUHAMMAD ASHRAFTARIQand IVORL. Pasiss
426
The specific activity of [75Se]selenite and [75Se]selenate selenium as a function of time in some of the individual organs is shown in Figs l(a, b) and 2(a, b),
respectively. Liver contains maximum activity at earlier periods in the experiment and was ten times as large as whole body activity for both the selenite and the selenate. The kidney content begins to exceed that of liver 24 h post-injection but continues to be less than the initial kidney content. The specific activities in the kidney after 24 h were 3.79 and 2.68 times the wholebody activity for selenite and selenate selenium, respectively. The lowest levels of activity were found in brain and muscle over the whole period of investigation. Blood, spleen, lungs and heart contents differ among each other by small factors and were random around the wholebody activity. The content of two selenium species in testicles was low in the beginning but became significant after a few days. Testicles accumulated higher concentrations of selenate than of selenite. The specific activity in kidney, spleen, blood and lungs were higher for selenite than for selenate after
24 h. The digestive system retained relatively more selenate than selenite. At the end of the study period, all tissues analyzed, except testes and brain, have higher selenite content as compared to selenate. Retention of selenium The retention of [” Selselenite and [” Selselenate after a single interaocular injection was monitored in blood and 10 tissue samples from animals sacrificed at fixed time intervals over a period of 31 days. The results are shown in Figs 3 and 4(a-d) for a few of the tissues investigated. Wholebody A rapid excretion of both selenite and selenate is observed in the first 24 h. The differences in the wholebody retention behavior become significant after 2 days with selenite content higher than the selenate. Nonetheless, Se(IV) and Se(M) excretion follow a similar pattern of rapid excretion followed by a slower phase. The excretion in the latter phase follows a first-order kinetic behavior with biological
Time dependent distribution of Se(V1) in normal mice LEGEND eZa Liver m Kidney U Whole Blood C!Z¶ Spleen C?Sl Testicles
Time (day) LEGEND tZZl Stomach
0.04
0.08
1.00
2.00 4.00 Time (day)
8.00
18.0
31.0
Fig 2. (a, b) The average content of [75Selselenate for 3 mice in a particular organ at different times after injection. Specific activity = cpm/g (organ&pm/g wholebody 1 h after injection.
Distribution of selenate and selenite selenium in mice
427 1: l -Liver-Se IV I 2: o-Liver-Se &A
1: .-Whole body,Se 2: o -Whole body,Se
3 -1.0
I
I
12.2
5.6
I
I
I
18.8
25.4
32.0
2)
-1.0
1
I
I
5.5
120
18.5
25.0
31.5
55
12.0
18 5
25.0
31.5
65
li.0
lb.5
2b.o
3i 5
Days Post Injection Fig. 3. Wholebody retention of selenite and selenate at different times after injection. Normalized to initial wholebody activity. Each point represents the mean body retention of 3 mice at the time of sacrifice. The individual data points were within 10%.
half-lives of 17 days for the two selenium species (Fig. 3). Liver
The excretion rate of the two selenium species in liver is very rapid in the initial phase where more than 80% activity is excreted within 1 day [Fig. 4(a)]. A slow change is observed in the next 15 days which follows another rapid excretion. After 30 days only 3-S% of initial dose is retained. The excretion does not follow the first-order kinetics and the concentration decreased to half the initial amount in approx. 16 days in the slower phase of the excretion.
3
-10
r
Kidney and stomach
The kidney retention was more uniform as compared to the liver throughout the experimental period with a lower content for selenate up to the end of the study [Fig. 4(b)]. In the second phase the concentration decreased by a factor of 2 in 11 and 12 days for selenite and selenate, respectively. The distribution of selenate and selenite in the gastrointestinal tract (stomach and the intestines only) exhibited a complementary characteristic for kidney. The selenite content in the stomach was lower than selenate, the case in kidney is opposite. Yet, the excretion half-lives of the stomach were around 9 days for both species.
1 _I
-1.0
Lungs, spleen and wholeblood
The retention in the lungs, spleen and wholeblood fluctuated and did not follow any specific pattern [Fig. 4(c)]. No rapid depletion phase was observed in these organs and the observation was common for both selenium species.
Jr-\ IS
Testicles
Rather than a depletion, an accumulation was observed in this organ for both chemical forms of selenium. After 30 days, 50% of selenite and 70% of initial selenate were still retained [Fig. 4(d)].
LO.0
15.5
21.0
20.5
Days Post Injection Fig. 4. (ad) Retention of selenite and selenate at different times after injection. Normalized to initial wholebody activity. Each point is the mean organ retention of 3 mice. The individual data points were within 10%.
MUHAMMADASHRAF TARIQand
428 Discussion
The general character of the distribution of intrinsic selenium in various tissues of C57L/J mouse (Table 1) is similar to the pattern observed in other studies reported in the literature (Behne and Wolters, 1983; Kinder et al., 1988; McConnell, 1941). The percent distribution, however, is different as shown in Table 2. This can, in part, be accounted for because the gastrointestinal tract and contents were also included as one of the organs in this study. This inclusion was necessary since the mechanism for quantitative absorption and simulation of the IV and VI oxidation states via the digestive tract are different (Korpela, 1988; Wolfram et al., 1986, 1988). The total content of selenium was highest in muscle, followed, in order, by liver, blood, kidney and gastrointestinal tract and its contents. Selenium content in the mouse kidney and testes differ, from that which has been reported for rats (Behne and Wolters, 1983; Hopkins et al., 1966). The distribution of [75Se]selenite or [“Selselenate 1 day after it is administered by injection does not follow the pattern of intrinsic selenium. The content in the blood is similar but the muscle pool contains one half when normal ingestion is compared. The liver, kidney and digestive system contents are higher, exhibiting the preferential retention of the selenium supplemented as oxyanions by injection. Although the initial specific activity was similar, the content of [“Se]selenite in the tissues analyzed was found to be higher than [75Selselenate throughout the experimental period [see Figs l(a, b) and 2(a, b) for comparison]. The distribution patterns of selenite and selenate are amazingly similar except for differences in excretory organs. It is the absolute concentration in a given tissue type that differs. The percentage of selenite retained by the kidney was always higher than that of selenate. The opposite was observed in the stomach. The specific activity of urine and feces was monitored around 24 h after injection. The activity of selenite was higher in urine and that of selenate in the feces. (Total content after an extended period is not available in this experiment.) It appears that more selenite is excreted through the kidney, while more selenate is retained by the stomach and the digestive system in the initial 24 h phase. The wholebody and organs follow an excretion pattern that exhibits two distinct rate dependencies for both of the radioactive selenium species. A rapid initial phase within the first 24 h and a slower phase after that [Figs 3 and 4(a-d)]. Similar behavior has been reported in other studies (Thorlacius-Ussing and Jensen, 1988). The excretion rate for organs in the slower phase may be first order if an uncertainty of f20% were included for each data point. However, the uncertainty among individual mice does not exceed + 10%. It therefore appears that the mechanism
IVORL. Pasrss
of organ excretion in the slower phase may be complex. The fluctuations in the elimination curves for various organs can be explained if selenium, in one chemical form, is deposited in the tissue while in another form it is released at a different rate. This is evident from the fact that wholebody excretion follows a first-order kinetic pattern for selenite and selenate (Fig. 3). Wholebody selenate retention was 13% higher than selenate 1 day after injection. After that the excretion of selenate was faster and the wholebody selenite specific activity was about 13% higher than that of the selenate after 8 days. However, the slopes of the two curves are similar (the rates are similar but the concentrations differ). The relative accumulation in testes was much higher in our experiment than reported for rats by others. This difference may arise from the very low dose of selenium administered in this study in an attempt to keep the physiology of the mouse unperturbed by not raising the concentration of the toxin (i.e. selenium). It is well known now that the distribution of selenium is dose-dependent (Hopkins et al., 1966). Two species seem to differ only in the initial phase. The higher retention of whole body selenate in the initial phase can be explained if the contents of the gastrointestinal tract remain within the mouse body for a longer period than urine. The percent distribution of radioactive selenate or selenite in the liver after 1 day is almost the same suggesting a common chemical form at this site of high metabolic activity for selenium. This in turn suggests that selenate is converted to selenite before being incorporated in protein. Because the content of selenite is always higher [Fig. 4(a)] it could be inferred that selenite has some direct pathway while selenate must be converted into selenite within the initial time phase following injection. It is also clear that not all of the selenate follows the selenite pathway. More detailed studies in the very early stage and sensitive in ciuo or in vitro speciation techniques are required to quantify these differences. Conclusions The specific metabolic chemistry of selenate differs from that of selenite. The apparent reduction to the plus IV state in the liver is but one indication of this difference in pathway. It is possible that many of the contradictory conclusions that appear in the literature have their basis in the fundamental chemical differences of the two oxyanions. It must be expected that SeO:- would follow the chemistry of sulfur (SO:-) while the thermodynamics of the oxidation reduction complex for SeO:- are significantly different than those of sulfite. Thus, oxidation state IV must follow a pathway independent of sulfur, and independent of the carrier effects of the sulfur oxyanions.
Distribution of selenate and selenite selenium in mice Acknowledgements-We
want to acknowledge the support from USPHS Grant 7RR0 7104. M. A. Taria wishes to thank the Government of Pakistan for its continuing support. Also support from Zeta International Ltd is gratefully acknowledged.
429
the female ICR mouse. Bull. envir. Contam. Toxicol. 40, 425.
Korpela H. (1988) Comparative effect of selenate and selenite on serum selenium concentration and glutathione peroxidase activity in selenium-depleted rats. Ann. Nutr. Metab. 32, 347.
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