C~~m.-3io~. ~~te~~o~, 12 (1976) 109-120 @ Elsevier Scientific Publishing Company, Amsterdam
- Printed in The Netherlands
BIQCHEMiCAL CHANGES IN RAT KIDNEY ON EXPOSURE TO ELEMENTAL MERCURY WAPOR: EFFECT ON BIOSYNTIIESIS OF METALLOTHIONEIN*
M.C. CHERIANa and T.W. CLARKSONb of Pharmacology and Toxicology, and b~e~rt~en~ of Radiation R~ophysics, The ~n~~~i~ of Rochester School of medicine, R~heste~, N.Y. 14642 (U.S,A.J
*Department Biology end
{Received February 2&h, 1975) (Revision received August 4th, 1975) (Accepted August&h, 1975)
SUMMARY Evrdence is presented that exposure of rats to elemental mercury vapor results in increased amounts of a me~othionein-like protein in kidney tissue but not in liver. After three or more daily exposures, each of 2 h duration, to elemental mercury vapor, more than 50% of the mercury in kidney tissue is bound to a protein having a molecular weight (mol. wt.) of about 10 000 as determined by Sephadex G-75 gel filtration chromatography. Cystine is incorporated into a 10 000 mol. wt. protein fraction from kidneys of rati which were injected with [U-‘*Cl] cystine after five daily 2-h exposures to mercury vapor. In contrast, no significant incorporation of [U-‘4C] cystine into this protein fraction was observed in kidneys of control rats or in livers of both control and mercury vapor-exposed rats. The in vivo incorporat.ion of logCd into the fraction followed the same pattern as that of [ 14C]cystine in rats injected with tracer doses of CdClz labeled with radioactive Io9Cd isotope. This 10 000 mol. wt. ~ro~in, newly synthesiz~ in response to repeated exposures to mercury vapor, exhibited identical properties to me~~o~ionein, namely in its subcellular localization, molecular weight, heat stability and isoelectric points. A significant incorporation of [ U-14CI-
;‘%ier is based on work performed under contract with the U.S. Energy Research and Development Administration at the University of Rochester Biomedical and Environmental Research Project and has been assigned Report No. UR-3490-816 and on work supported by Grant No, POl-ES01059 from the National Institute of Health. By acceptance of this a&Me, the publisher and/or recipient acknowledges the U.S. ~overnment’s right to retain a nonexciusive, royalty-free license in and to any copyright covering this paper. aPresent address: Department of Pathology, University of Western Ontario, London, Ontario, Canada.
109
cystine into this protein int rat kidney alone on exposure to mercury vapor confirms its induced biosynthesis in the kidney, INTRODUCTION
The induced synthesis of me~~o~ionein, a low molecular weight cytoplasmic protein with high content of cystine, has been reported in rabbit and rat liver in response to administration of cadmium [ 1.2). Rmmt study [$I] shows that repeated injection of mercuric chloride in rats can also induce the synthesis of metallothionein in kidney but not in liver. Me~lo~ione~ has high affinity for heavy metals like c~rni~ and rne~u~ and its p~~~~ role in the heavy metal toxicity has been speculated [4]. Inhalation of elemental mercury vapor has been an occupationaI health hazard to man since antiquity [5], Thus it seemed important to develop rat model experiments to study the biochemical effects of long term exposure to metallic mercu~ vapor. Previous studies have shown that element mercury vapor (Hg’) is rapidly oxidized to divalent ionic mercury (Hg*) on exposure to red blood cells in vitro and following in vivo uptake by animals [6,7]. Thus the possibility arises that exposure to mercury may lead ta increased tissue levels of metallothionein. These experiments were undertaken to check this possibility in rats repeatedly exposed to mercury vapor. MATERIALS AND METHODS
Radioactive *03Hglabeled mercuric chloride (spec. activity 6 mCi/mg H ‘09CdG12 (carrier free) and uniformly labeled [‘4C]cystine (spec. a&iv&y 250 mCi/~ole) were obtained from New Engl~d Nuclei Co~om~on. Female Sprague-Dawley rats weighing 156 to 260 g were used in the experiments. Rats were exposed to mercury vapor in a desiccator as described previously [ES].The source of mercury vapor was either elemental mercury in a closed system through which a constant air flow (0.8 l/min) was maintained or a spirometer filled with radioactive ‘O~H~rne~~ vapor. The radioactive *03H~rne~u~ vapqr was prepared from 2031=igC12 by the method of Clarkson and Greenwood [9]. The total mercury in liver and kidney homogenates and in the subcellular fractions was measured by the method of Magos and Clarkson [lo] after different exposures to stable mercury vapor, The exposures of rats to elements rne~u~ vapor were as fo~o~. Three rats were exposed simultaneously in the same desiccator. Each exposure period was always for 2 h and only one exposure was carried out each day. The number of days the animals were exposed varied from 1 to 8, depending upon the type of experiments as described in the app~p~a~ figure legends and tables. For incorporation studies of logCdinto protein fractions of liver and kidney, the rats were injected (i.p.) with CdC12 (1 mg Cd/kg) labeled wih u?X 110
to mercury vapor (non-~ioactive) for Cal 7 conduct day following the cadmium dose, the animals received another exposure to mercury vapor. These animals along with controls (not exposed to vapor) were sacrificed 3 h after the inje&bn of
00 ~~~~
cdcl~. To study the effect of mercury vapor on incorporation
of [U-*4C]cystine into kidney and liver protein, rats were exposed to non-radioactive mercury vapor for 7 days. They received an iq. injection of [ Ut4C] cystine (20 PC%/ rat) followed by @X~CBWQ to the vapor and were sacrificed 24 h later, In this selected for the iylcorporation of [ 14C] cystine because of’ the low turnover of this p&otein proteinase. A similar time period was used by ~0~~~~ et d. 13) and by Squibb and Cousins [12] in their studies of rn~~o~~one~ synthesis. The effect of n the incorporation of cystine into proteins of liver and kidney by injection of [Uv’4C] cystine (20 pCi/rat) into (non-cadmium treated) and exposed animals. The latter received a on of CdCl2 (0.25 mg Cd/kg) on each of 2 days prior to the sacrificed by bleeding, liver and kidneys were removed and ized in a Pot~~Elvehjem teflon homogenizer in 0.25 M sucrose(~s-KCl~~l~) buffer pH 7.4,O.l M Tris, 0.025 M KCI and 0.005 M The tissue homogena~s (20%) were subf~~ona~d into nuclear, ondrial, microsomal and supematant fractions (105 000 g for 30 min) by differential centrifugation using standard procedure 1131. The supernatant fractions from both liver and kidney were fractionated on a calibrated Sephadex G-75 column (0.9 X 60 cm), which was calibrated with blue dextran (200 000), aldolase (158 000), ovalbumin (45 000), chymotrypsinogen (25 000), ribonuclease (13 700) and bacitracin (1400). Sodium phosphate buffer pH 8.6, 0.1 M with 0.2 M sodium chlotide was used as elution buffer and the flow rate was 12 ml/h. The UV absorption at 254 and 280 nm were monitored in a LKB UV Cord III and 1 ml fractions were collected in a Ul~Back fraction collector. The fractions were corned for *03Hgor “‘Cd in a Packard g~ma aviation counter with counting ef~ciency of 36% % in a Packard liquid scintillation counter respectively and for and ing 10 ml of premix Aquasol from New England Nuclear Corporaafte tion with a counting efficiency of more than 90%. Tissues and subcellular fractions were solubilized in 1 ml of Protosol (New England Nuclear Corp.) and neutralized with 61 ml of acetic acid before adding Aquasol mix. Protein was determined by the method of Lowry et al. [ 14 3. Me~lo~hion~in was isolated from kidneys of rats by the method developed ectric focusing was carried out by the method in this l~bo~~~ [ 16 6], modified as follows. The dense and light of ~a~ark and Ves with 2 ml and 0.5 ml of ~pholine pH 3-6 solutions were mix SUC festively, Anode solution was prepared by dissolving 12 g of sucrose in 14 ml of demine~i~~ water and 0.2 ml of orthophosphoric acid (80%). 111
Sample (1 ml) was mixed with dense sucrose solution (1 m to the middle of sucrose gradient in LKB 110columnwitha The cathode solution consisted of 1 0.2. ml of monoethanolamine ad gradient. During the first 6 h or‘ elec the column and the voltage was slowly acurrent less than 2 mA, After 48 h, the e fractions were collected, counted fo monitored and pH of each fraction was Cadmium was estimated in tissue homo tion of approx. by Pulido et al. [!7] and had a liiit of weight of tissue. RESULTS
The data on deposition of mercury after repea mercury vapor confirm the finding by Rothstein deposition is much greater than deposition in the live ng Hg/g tissue protein, the concentration of mercury in the liver by at least a factor of 60. The am
TABLE I ACCUMULATION OF MERCURY IN RAT LIVER AND KIDNEY AFTER R EXPOSURE TO MERCURY VAPORa Number of Hg vapor exposures 1 2 3 4
Liver
Kidney
ng Hglmtt protein
total Hg MB
ng PrsIlmgt protein
total Dig lrgt
0.7 1.6 2.4 2.0
1.4 2.6 3.2 3.6
2:: 870 343
19, Cal I13 I2I
a Rats were exposed to stable metallic mercury vaptx Co AND METHODS. They were sacrificed 24 h after the is the amount of mercury per mg protein in tissue. “To resent in the whole organ, i: The average air concentration of mercury vapor was calcuhcted from deposited in the kidneys using metabolic data from Rothskin and rats repeatedly exposed to 100% of the labeled vnpor half-time of 20 days.
112
D
QN QP
CtJRY 1-NRAT KIDNEY AFTER EXPOSURE
of Homogenate 1
218
loo 22
52
100 27
1
11
6.6
5.2
11
10
5.7
4.7
112
127
57
58
figure is the mean for three ani-
preparedseparately from each ani-
the first 3 days and, thereafter, more
in air were not measured directly but were estimated as described in the legend of Table I. The on during expure was 4.1 mg Hg/m3. in alI subcellular fractions following eight daily e u&g enzyme Mar particles by II give only an ar distribution. However, the supematant of other suhcellular particles contained the the kidney (approx. 57 to 58%). soluble fraction on Sephadex G-75 colthe mercury in this fraction is bound . 1). This protein fraction also showed shared with metallokidney homogenate e atomic absorption suggest that mercury, to the vapor, binds to a t.kx similar to those of metallothionein. ration of ‘ooed into the 10 000 mol. wt. fraction of sint to the induction of metallothionein by repeated 0 mercury vapor, ular weight protein fraction as tionation of kidney supematant. Only a 10 000 mol. wt. fractions. No significant
113
FRAtTlON NUMBERS
Fig. 1. Rats were exposed 2 h per day to non-radioactjve mercury vapot for 7 consecutive days. On the 8 day, they were exposed to mercury vapor Iaheled with the *Hg isotoplt and sacrificed 24 h later. The 105 000 g supematant of the kidney homogenate was heated at 85O for 2 min, centrifuged and chromatographed on a G-75 Sephadex column. (For details, see MATERIALS AND METHODS. About 92% of radioactivity was recovered from the column. *o--o, Counts z”3Hg~mintfraction; - - - - - -, absorbance at 284 nm.
change in absorbance (254 nm) in the 10 000 mol. wt. fractions could be seen. In contrast, most of the cadmium in kidney supernatant was associated with the 10 000 mol. wt. fractions and a significant absorption maximum at (254 nm) was seen in animals repeatedly exposed to mercury vapor. Sephadex G-15 fracti~na~on of supe~atant from liver {not shown in Fig, 2) indica~ negligible Cd incorporation and insignificant absorbance (254 nm) changes
in the 10 000 mol. wt. fractions in both control and mercury vapor-expos animals. In liver supernatant, as observed in kidney supe~a~t of eontro animals, most of the cadmium was associated with the high mol~ul~ alit protein fractions. The data in Table III indicate that repeated exposure to mercury vapor increases the amount of radioactivity found in the supernatant fractions of the kidneys in rats given [U-14C] cystine. The radioactivity in the whole homogenate of the kidneys exhibited f small increase for the exposed rats, but the increase was not statistic~Iy si~ific~t. However, when the supernatant fraction was tested, a highly significant increase in inco~oration w observed in mercury vapor-exposed animals (1247 cpm/mg protein in control compared 1679 in exposed animals). Exposure to mercury vapor had no signific~t effect on levels of r4C radioactivity in the superna~nt fractions from livers of the same dirndl. The incorporation of [ 14C1 eystine radioactivity into total liver proteins was slightly but significantly depressed by mercury vapor ex.posure. In view of the findings in Table III, it was decided to follow the incorporation of l4 C in Sephadex G-75 fractions of liver and kidney swpernatant. The Sephadex G-75 fractions from kidneys of non-exposed rats indica~ only slight inco~oration of i4C and no detectable change in absorbance in 10 006 mol. wt. region (I?ig. 3). The Sephadex pattern for rats repeatedly 114
FRACION NUMRfRS
Fig. 2. Gel filtration of kidney supematants on Sephadex G-75 column from control rat and rats exposed to mercury vapor for 8 days. Rats were injected with l@%lJdC12 (i.p.) 3 h before sacrifice. 98% of radioactivity was recovered from the column. . - 1 -a, WZd radi~ctivity ; - - - - f -, absorbance at 254 nm. Fig. 3. Sephadex G-75 fractionation of kidney supernatants from control rats, rats exposed to mercury vapor for 8 days and rats injected with CdCl2 (i.p.) for 2 days. [ W]Cystine (28 rCi. i-v.) was injected 24 h before sacrifice. 90% of radioactivity was recovered from the column. . - +- a, [ W$] cystine radioactivity; - - - - - -. absarbance at 254 nm.
exposed to mercury vapor indicates a large increase of 14Cradioac.Lvity and a si~iiic~t abso~tion rn~~urn at 254 nm in the 10 000 mol. wt. fractions. reagent of animals with CdC12, a procedure known to induce metallot~ionein [2,3], produced Sephadex chromatographic patterns similar to those seen m inercury-exposed rats except that incorporation of 14C radioactivity in the “metallothionein” fractions was less in kidney supernatant. The da& in Fig. 4 indicate that pretreatment of rats with CdClz increases levels of mstaUothionein-like protein whereas repeated exposure to mercury has no effect. In rats pretreated with CdClz both the 14C radioactivity and the absorbance at 254 nm increased in Sephadex fractions corresponding to the 10 066 mol. wt. region. Repeated exposure to mercury vapor produced table effect on % radioactivity and the absorbance at 254 nm in the IO ~~~ mol. wt. axon. Fu~er eh~a~~~zation of the newly synthesized protein in rat kidney after exposure to mercury vapor was achieved by comparing its heat stability
115
TABLE HI IN WV0 INCORPORATION OF [ tJ-14C] CYSTINE INTO LIVER AND KIDNEY OF CONTROL RATS AND RATS EXPOSED TO MERCURY VAPOR Rats were injected with 20 /Zi of [ 14C Jcystine (i.v.) 24 h prior to sacrifice. The experimental rata were exposed to stable metalliz mercury vapor for 7 daye before being injected with [ 14C Jcystine and were again exposed to mercury vapor fo., 2 h before sacrifice 24 h later. The results are Mean f SE. of three separate experiments. Three rats were used in each experiment. cpm/mg Protein
Liver Control Hg-Exposed Kidney Control Hg-Exposed --
Homogenate
Supernetant
816i 11 728 + 22
1228 * 42 1238 * 44
1057 f 35 1078 * 61
1247 * 51 1679 * 47
MfRC”RY VAWR
WOStD
CADMWM INJfCIfD
M FRACTION
40 WMBERS
Fig. 4. Sephadex G-76 fractionation of liver supernatants from control rats, rats expoeed to mercury vapor for 8 days and rats injected with CdCla for 2 days. [ i4C] Cystine (20 r Ci, i.v.) was injected 24 h before sacrifice. . - 1 - a, [i4C]cystine radioactivity; ----=*t absorbance at 254 nm.
116
Fii. 5. Get filtration control
on Sephadex
rats and rate exposed
with [ *%]cystine
G-75 columns of heated kidney supernatants
from
to metallic mercury vapor for 8 days. Rats were injected
(20 rCi, i.v.) 24 h before sacrifice. 105 000 g su~rnatant
was prepared
from kidney, heated at HP for 2 min and centrifuged at 10 000 g for 10 min. . - e- *, [ W]cyatine radioactivity; - - - - - -, absorbance 254 nm.
and isoelectric point with metallotbionein [ 15 ] . The data in Fig. 5 indicate the effect of heating the kidney supe~a~t from control and exposed animals. Heating is known to cause precipitation of high molecular weight proteins but to leave metallothionein unaffected [20]. Comparing the data in Fig. 5 with those in Fig. 3, it is apparent that heating the *4C-labeled supemataitt from kidneys of exposed animals results in loss of ~dioac~~~ specifically from the high molecular weight protein peak and complete recovery of r~~oactivity in the me~lothionein region.
0
IO
M
30
40
so
6’1
70
II0
1x3
FRACTION NUMBERS
Fig. 6. The profiles of WHg radioactivity (a - * - .) and absorbance at 254 nm (- - - - - -1 following isoelectric focusing of the 10 000 mol. wt. fraction from kidneys of rats repeatedly exposed to etemenlal mercury vapor. (For details, see MATERIALS AND METHODS.) The pii of the fraction is indicated by the continuous line.
117
A partial pu~fi~ation of the 10 000 mol. wt. proteins was ~~~rnp~ from rat kidney after repeated exposure to mercury vapor fo~owing the methods of Cherian /15]. The 10 000 mol. wt. fraction from Sephadex G-75 filtration was subjected to isoelectric focusing using ampholines, pH 3-6. Two protein peaks and corresponding mercury peaks were observed (Fig. 6), the isoelectric points being at pH 4.5 and pH 4.9. About 80% of the total radioactivity ap plied on the column can be recovered in the two main mercury peaks. These results are similar to thclise reported previously for isoelectric focusing of metallothionein induced in rat liver on pretreatment with cadmium salts [1!5] _ In a recent report, Nordberg et al. (211 have observed different isoelectric points (above pH 11) for mercury binding proteins in rabbit kidney after a~~ist~tion of mercuric chloride. DISCVSSION
The experimental evidence presented in this report indicates that repeated exposure of rats to elemental mercury vapor leads to the induction of metallothionein-like protein if not metallothionein itself. The size of induction is probably the kidney but confirmation would require in vitro experiments. The evidence may be briefly summarized as follows: (I) Repeated exposure of rats to mercury leads to a shift in the subcellular dis~bution of mercury in kidney tissue with most of the mercury being deposi~d in the soluble fraction. Further frac~ona~ons of the 105 OOOg ~pema~nt indicates that more mercury is bound to a 10000 mol. wt. fraction in rats having mtiltiple exposure as compared to animals reccPiving a single exposure to mercury vapor (Cherian, unpublished data). (2) Repeated exposure to mercury vapor results in an increased absorption at 254 nm in the 10 000 mol. wt. fraction from kidney supematant - a result similar to that obtained when metallothionein is induced by CdC12. (3) Repeated exposures to mercury vapor result in changes in the subcellular distribution of injected tracer doses of *09Cd, favoring its binding to the 10 000 mol. wt. fraction. (4) hollowing the approach of Piotro~ki et al. [3] the rate of in vivo ~co~oration of [UJ4C] cystine was taken as a measure of the biosyn~es~ of ‘~me~lo~ionein-like” proteins. Repeated exposure to mercury vapor enhanced the biosynthesis in kidney, but not in liver, (5) The similarity of the newly synthesized protein with metallothionein is also evident from its heat stability and its isoelectric points. The diffctrence in isoelectric points of proteins in the present study and that of NordMrg et al. [Zl] in rabbit kidney is not clear. However, it should be mentioned that pI more than 10 is usually associated with proteins like histones. (6) That the kidney is the probable site of synthesis is indicated ?,y the findings that the newly synthesized protein can be detected in kidney tissue but not in liver, These results confirm the findings of Sapota et al. 1221 on studies of rats repeatedly exposed to mercury vapor. These authors reported similar results 118
in pregnant rata but fetal levels of metahothionein were not increased by maternal exposure to mercury vapor. The effecb of exposure to mercury vapor are similar to those reported by Piotrowski et al. [3], in rats repeatedly dosed with mercuric chloride. This similarity ie not surprising since elemental mercury vapor is rapidly converted to ionic mercury in animal tissue and, as with HgCl*, accumulates preferentially ig the kidneys [ 181. The reason that exposure to mercury vapor does not lead to the induction of pro&An in the liver is probably due to the much lower levels of mercury in the liver, as is disc by ~o~~ki et al., [3] in expiring their similar with repeated doses of inorganic salts of mercury. find gnificance of these findings to human exposure to elemental mercury T vapor deserves 91brief comment. These experiments were made at high vapor ~oncent~~ons (about 4 mg Hg/m3). occupations exposures are at least an order of m itude lower than this. However, exposure times in our experiments were brief (2 h), and the animals were exposed for not more than 8 days. The induction of metallothionein in kidney tissue and the binding of mercury to this protein may be one explanation for the persistence of mercury in utile long after cessation of occupational exposure [23] and for the reports of human tolerance to mercury vapor in long-tea occupa~on~ exposure [ 241.
We wish to ~~k3udi assistance.
Allen and Ron Vander Mallie for excellent technical
REFERENCES
1 M. Piscator, On cadmium in normal human kidneys together with a report on the isolation of metallothionein from livers of cadmium exposed rabbits, Nerd. Hyg. T., 45 (1964) 76. 2 EA. Shaikh and 0.J. Lucis, Induttion of cadmium binding proteins, Fed. Proc., 99 (1976) 298. J.K. Piotrowski, B, ~ojanowska, J.M. Wisniewska-Kny~l and W. Boianowska, Mercury binding in the kidney and liver of rata repeatedly exposed to mercuric chloride: Induction of metallothionein by mercury and cadmium, Toxicol. Appl. Pharmacol., 27 (1974) 11. G.F. Nordbarg, Effects of acute and chronic cadium exposure on the testicles of mice, Environ. Physiol., 1 (1971) 171. D. Hunter, The Ancient Metals in Diseases of Occupations, Brown, London, 1969. T.W. Clarkson, T. Gatxy and C. Dayton, Studies on the equilibrium of mercury vapor with blood, ABC Research and Development, Report UR582,1961. L. Mngos, Mercury-blood interaction and mercury uptake by the brain after vapor exposure, Environ, Res., 1 (1967) 323. Y. Sugata, T.W. Clarkson and L. Magos, An apparatus to expose small animals to radjoaetive mercury vapor, Brit. J. Ind. Med., {1975) (in press).
119
9 T.W. Clarkson and M. Greenwood, Selective determination of inorganic mercury in the presence of organomercurial compounds in biological material, Anal. Biochem., 37 (1970) 236. 16 L. Magos and T.W. Clarkson, Atomic abortion determination of total, inorganic and organic mercury in blood, J. Assoc. Offic. Anal. Chem., 55 (1972) 966. 11 G.F. Nordberg, Cadmium metabolism and toxicity, Environ. Physiol. Biochem., 2 (1972) 7. 12 K.S. Squibb and R.J. Cousins, Control of cadmium binding protein synthesis in rat liver, Environ. Physiol. Biochem., 4 (1974) 24. 13 G.H. Hogeboom, Fractionation of cell components of animal tiawes, in S.P. Colowick and N.O. Kaplan (Eds.), Methods in Enzymology, Vol. I, Academic Press, New York, 1955, pp. 16-18. 14 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the Folia phenol reagent, J. Biol. Chem., 193 (1951) 265. 15 M.G. Cherian, Isolation and purification of cadmium binding proteins from rat liver, Biochem. Biophys. Res. Commun., 61(1974) 920. 16 T. Flatmark and 0. Vesterberg, On the heterogeneity of beef heart cytochrome C, IV. isoelectric f~~tionation by electroy& in a natural pH gradient, Acta C&em. Stand., 20 (1966) 1497. 17 P. Pulido, K. Fuwa and 8.L Vallee, &termination of cadmium in biological material by atomic absorption spectrophotometry, Anal. Biochem., 14 (1966) 393. 18 A. Rothstein and A. Hayes, The turnover of mercury in rata exposed repeatedly to inhalation of vapor, Health Phys., 10 (1964) 1099. 19 T. Norseth, Studies on intracellular distribution of mercury, in M.W. Miller and G.G. Berg (Eds.), Chemical Fallout: Current Research on Persistent Pesticides, Thomas, Springfield, ill., 1969. 20 M. Webb, Binding of cadmium ions by rat liver and kidney, Biochem. Pharmacol., 21 (1972) 2751. 21 M. Nordberg, B. Trojanowska and G,F. Nordberg, Studies on metal binding proteins of low molecular weight from renal tissue of rabbits exposed to cadmium or mercury, Environ. Physiol. Biochem,, 4 (1974) 149. 22 A. Sapota, J.K. Piotrowski and B. Baranski, Level of met~lothionejn in the fetuses and tissues of pregnant rats exposed to mercury vapor, Med. Pracy, XXV (1974) 129 (In Polish). 23 L.J. Goldwater and A. Nicolan, Absorption and excretion of mercury in man, IX. Persistence of mercury in blood and urine following cessation of exposure, Arch, Environ. Health, 12 (1966) 196.
- Printed in The Netherlands
BIQCHEMiCAL CHANGES IN RAT KIDNEY ON EXPOSURE TO ELEMENTAL MERCURY WAPOR: EFFECT ON BIOSYNTIIESIS OF METALLOTHIONEIN*
M.C. CHERIANa and T.W. CLARKSONb of Pharmacology and Toxicology, and b~e~rt~en~ of Radiation R~ophysics, The ~n~~~i~ of Rochester School of medicine, R~heste~, N.Y. 14642 (U.S,A.J
*Department Biology end
{Received February 2&h, 1975) (Revision received August 4th, 1975) (Accepted August&h, 1975)
SUMMARY Evrdence is presented that exposure of rats to elemental mercury vapor results in increased amounts of a me~othionein-like protein in kidney tissue but not in liver. After three or more daily exposures, each of 2 h duration, to elemental mercury vapor, more than 50% of the mercury in kidney tissue is bound to a protein having a molecular weight (mol. wt.) of about 10 000 as determined by Sephadex G-75 gel filtration chromatography. Cystine is incorporated into a 10 000 mol. wt. protein fraction from kidneys of rati which were injected with [U-‘*Cl] cystine after five daily 2-h exposures to mercury vapor. In contrast, no significant incorporation of [U-‘4C] cystine into this protein fraction was observed in kidneys of control rats or in livers of both control and mercury vapor-exposed rats. The in vivo incorporat.ion of logCd into the fraction followed the same pattern as that of [ 14C]cystine in rats injected with tracer doses of CdClz labeled with radioactive Io9Cd isotope. This 10 000 mol. wt. ~ro~in, newly synthesiz~ in response to repeated exposures to mercury vapor, exhibited identical properties to me~~o~ionein, namely in its subcellular localization, molecular weight, heat stability and isoelectric points. A significant incorporation of [ U-14CI-
;‘%ier is based on work performed under contract with the U.S. Energy Research and Development Administration at the University of Rochester Biomedical and Environmental Research Project and has been assigned Report No. UR-3490-816 and on work supported by Grant No, POl-ES01059 from the National Institute of Health. By acceptance of this a&Me, the publisher and/or recipient acknowledges the U.S. ~overnment’s right to retain a nonexciusive, royalty-free license in and to any copyright covering this paper. aPresent address: Department of Pathology, University of Western Ontario, London, Ontario, Canada.
109
cystine into this protein int rat kidney alone on exposure to mercury vapor confirms its induced biosynthesis in the kidney, INTRODUCTION
The induced synthesis of me~~o~ionein, a low molecular weight cytoplasmic protein with high content of cystine, has been reported in rabbit and rat liver in response to administration of cadmium [ 1.2). Rmmt study [$I] shows that repeated injection of mercuric chloride in rats can also induce the synthesis of metallothionein in kidney but not in liver. Me~lo~ione~ has high affinity for heavy metals like c~rni~ and rne~u~ and its p~~~~ role in the heavy metal toxicity has been speculated [4]. Inhalation of elemental mercury vapor has been an occupationaI health hazard to man since antiquity [5], Thus it seemed important to develop rat model experiments to study the biochemical effects of long term exposure to metallic mercu~ vapor. Previous studies have shown that element mercury vapor (Hg’) is rapidly oxidized to divalent ionic mercury (Hg*) on exposure to red blood cells in vitro and following in vivo uptake by animals [6,7]. Thus the possibility arises that exposure to mercury may lead ta increased tissue levels of metallothionein. These experiments were undertaken to check this possibility in rats repeatedly exposed to mercury vapor. MATERIALS AND METHODS
Radioactive *03Hglabeled mercuric chloride (spec. activity 6 mCi/mg H ‘09CdG12 (carrier free) and uniformly labeled [‘4C]cystine (spec. a&iv&y 250 mCi/~ole) were obtained from New Engl~d Nuclei Co~om~on. Female Sprague-Dawley rats weighing 156 to 260 g were used in the experiments. Rats were exposed to mercury vapor in a desiccator as described previously [ES].The source of mercury vapor was either elemental mercury in a closed system through which a constant air flow (0.8 l/min) was maintained or a spirometer filled with radioactive ‘O~H~rne~~ vapor. The radioactive *03H~rne~u~ vapqr was prepared from 2031=igC12 by the method of Clarkson and Greenwood [9]. The total mercury in liver and kidney homogenates and in the subcellular fractions was measured by the method of Magos and Clarkson [lo] after different exposures to stable mercury vapor, The exposures of rats to elements rne~u~ vapor were as fo~o~. Three rats were exposed simultaneously in the same desiccator. Each exposure period was always for 2 h and only one exposure was carried out each day. The number of days the animals were exposed varied from 1 to 8, depending upon the type of experiments as described in the app~p~a~ figure legends and tables. For incorporation studies of logCdinto protein fractions of liver and kidney, the rats were injected (i.p.) with CdC12 (1 mg Cd/kg) labeled wih u?X 110
to mercury vapor (non-~ioactive) for Cal 7 conduct day following the cadmium dose, the animals received another exposure to mercury vapor. These animals along with controls (not exposed to vapor) were sacrificed 3 h after the inje&bn of
00 ~~~~
cdcl~. To study the effect of mercury vapor on incorporation
of [U-*4C]cystine into kidney and liver protein, rats were exposed to non-radioactive mercury vapor for 7 days. They received an iq. injection of [ Ut4C] cystine (20 PC%/ rat) followed by @X~CBWQ to the vapor and were sacrificed 24 h later, In this selected for the iylcorporation of [ 14C] cystine because of’ the low turnover of this p&otein proteinase. A similar time period was used by ~0~~~~ et d. 13) and by Squibb and Cousins [12] in their studies of rn~~o~~one~ synthesis. The effect of n the incorporation of cystine into proteins of liver and kidney by injection of [Uv’4C] cystine (20 pCi/rat) into (non-cadmium treated) and exposed animals. The latter received a on of CdCl2 (0.25 mg Cd/kg) on each of 2 days prior to the sacrificed by bleeding, liver and kidneys were removed and ized in a Pot~~Elvehjem teflon homogenizer in 0.25 M sucrose(~s-KCl~~l~) buffer pH 7.4,O.l M Tris, 0.025 M KCI and 0.005 M The tissue homogena~s (20%) were subf~~ona~d into nuclear, ondrial, microsomal and supematant fractions (105 000 g for 30 min) by differential centrifugation using standard procedure 1131. The supernatant fractions from both liver and kidney were fractionated on a calibrated Sephadex G-75 column (0.9 X 60 cm), which was calibrated with blue dextran (200 000), aldolase (158 000), ovalbumin (45 000), chymotrypsinogen (25 000), ribonuclease (13 700) and bacitracin (1400). Sodium phosphate buffer pH 8.6, 0.1 M with 0.2 M sodium chlotide was used as elution buffer and the flow rate was 12 ml/h. The UV absorption at 254 and 280 nm were monitored in a LKB UV Cord III and 1 ml fractions were collected in a Ul~Back fraction collector. The fractions were corned for *03Hgor “‘Cd in a Packard g~ma aviation counter with counting ef~ciency of 36% % in a Packard liquid scintillation counter respectively and for and ing 10 ml of premix Aquasol from New England Nuclear Corporaafte tion with a counting efficiency of more than 90%. Tissues and subcellular fractions were solubilized in 1 ml of Protosol (New England Nuclear Corp.) and neutralized with 61 ml of acetic acid before adding Aquasol mix. Protein was determined by the method of Lowry et al. [ 14 3. Me~lo~hion~in was isolated from kidneys of rats by the method developed ectric focusing was carried out by the method in this l~bo~~~ [ 16 6], modified as follows. The dense and light of ~a~ark and Ves with 2 ml and 0.5 ml of ~pholine pH 3-6 solutions were mix SUC festively, Anode solution was prepared by dissolving 12 g of sucrose in 14 ml of demine~i~~ water and 0.2 ml of orthophosphoric acid (80%). 111
Sample (1 ml) was mixed with dense sucrose solution (1 m to the middle of sucrose gradient in LKB 110columnwitha The cathode solution consisted of 1 0.2. ml of monoethanolamine ad gradient. During the first 6 h or‘ elec the column and the voltage was slowly acurrent less than 2 mA, After 48 h, the e fractions were collected, counted fo monitored and pH of each fraction was Cadmium was estimated in tissue homo tion of approx. by Pulido et al. [!7] and had a liiit of weight of tissue. RESULTS
The data on deposition of mercury after repea mercury vapor confirm the finding by Rothstein deposition is much greater than deposition in the live ng Hg/g tissue protein, the concentration of mercury in the liver by at least a factor of 60. The am
TABLE I ACCUMULATION OF MERCURY IN RAT LIVER AND KIDNEY AFTER R EXPOSURE TO MERCURY VAPORa Number of Hg vapor exposures 1 2 3 4
Liver
Kidney
ng Hglmtt protein
total Hg MB
ng PrsIlmgt protein
total Dig lrgt
0.7 1.6 2.4 2.0
1.4 2.6 3.2 3.6
2:: 870 343
19, Cal I13 I2I
a Rats were exposed to stable metallic mercury vaptx Co AND METHODS. They were sacrificed 24 h after the is the amount of mercury per mg protein in tissue. “To resent in the whole organ, i: The average air concentration of mercury vapor was calcuhcted from deposited in the kidneys using metabolic data from Rothskin and rats repeatedly exposed to 100% of the labeled vnpor half-time of 20 days.
112
D
QN QP
CtJRY 1-NRAT KIDNEY AFTER EXPOSURE
of Homogenate 1
218
loo 22
52
100 27
1
11
6.6
5.2
11
10
5.7
4.7
112
127
57
58
figure is the mean for three ani-
preparedseparately from each ani-
the first 3 days and, thereafter, more
in air were not measured directly but were estimated as described in the legend of Table I. The on during expure was 4.1 mg Hg/m3. in alI subcellular fractions following eight daily e u&g enzyme Mar particles by II give only an ar distribution. However, the supematant of other suhcellular particles contained the the kidney (approx. 57 to 58%). soluble fraction on Sephadex G-75 colthe mercury in this fraction is bound . 1). This protein fraction also showed shared with metallokidney homogenate e atomic absorption suggest that mercury, to the vapor, binds to a t.kx similar to those of metallothionein. ration of ‘ooed into the 10 000 mol. wt. fraction of sint to the induction of metallothionein by repeated 0 mercury vapor, ular weight protein fraction as tionation of kidney supematant. Only a 10 000 mol. wt. fractions. No significant
113
FRAtTlON NUMBERS
Fig. 1. Rats were exposed 2 h per day to non-radioactjve mercury vapot for 7 consecutive days. On the 8 day, they were exposed to mercury vapor Iaheled with the *Hg isotoplt and sacrificed 24 h later. The 105 000 g supematant of the kidney homogenate was heated at 85O for 2 min, centrifuged and chromatographed on a G-75 Sephadex column. (For details, see MATERIALS AND METHODS. About 92% of radioactivity was recovered from the column. *o--o, Counts z”3Hg~mintfraction; - - - - - -, absorbance at 284 nm.
change in absorbance (254 nm) in the 10 000 mol. wt. fractions could be seen. In contrast, most of the cadmium in kidney supernatant was associated with the 10 000 mol. wt. fractions and a significant absorption maximum at (254 nm) was seen in animals repeatedly exposed to mercury vapor. Sephadex G-15 fracti~na~on of supe~atant from liver {not shown in Fig, 2) indica~ negligible Cd incorporation and insignificant absorbance (254 nm) changes
in the 10 000 mol. wt. fractions in both control and mercury vapor-expos animals. In liver supernatant, as observed in kidney supe~a~t of eontro animals, most of the cadmium was associated with the high mol~ul~ alit protein fractions. The data in Table III indicate that repeated exposure to mercury vapor increases the amount of radioactivity found in the supernatant fractions of the kidneys in rats given [U-14C] cystine. The radioactivity in the whole homogenate of the kidneys exhibited f small increase for the exposed rats, but the increase was not statistic~Iy si~ific~t. However, when the supernatant fraction was tested, a highly significant increase in inco~oration w observed in mercury vapor-exposed animals (1247 cpm/mg protein in control compared 1679 in exposed animals). Exposure to mercury vapor had no signific~t effect on levels of r4C radioactivity in the superna~nt fractions from livers of the same dirndl. The incorporation of [ 14C1 eystine radioactivity into total liver proteins was slightly but significantly depressed by mercury vapor ex.posure. In view of the findings in Table III, it was decided to follow the incorporation of l4 C in Sephadex G-75 fractions of liver and kidney swpernatant. The Sephadex G-75 fractions from kidneys of non-exposed rats indica~ only slight inco~oration of i4C and no detectable change in absorbance in 10 006 mol. wt. region (I?ig. 3). The Sephadex pattern for rats repeatedly 114
FRACION NUMRfRS
Fig. 2. Gel filtration of kidney supematants on Sephadex G-75 column from control rat and rats exposed to mercury vapor for 8 days. Rats were injected with l@%lJdC12 (i.p.) 3 h before sacrifice. 98% of radioactivity was recovered from the column. . - 1 -a, WZd radi~ctivity ; - - - - f -, absorbance at 254 nm. Fig. 3. Sephadex G-75 fractionation of kidney supernatants from control rats, rats exposed to mercury vapor for 8 days and rats injected with CdCl2 (i.p.) for 2 days. [ W]Cystine (28 rCi. i-v.) was injected 24 h before sacrifice. 90% of radioactivity was recovered from the column. . - +- a, [ W$] cystine radioactivity; - - - - - -. absarbance at 254 nm.
exposed to mercury vapor indicates a large increase of 14Cradioac.Lvity and a si~iiic~t abso~tion rn~~urn at 254 nm in the 10 000 mol. wt. fractions. reagent of animals with CdC12, a procedure known to induce metallot~ionein [2,3], produced Sephadex chromatographic patterns similar to those seen m inercury-exposed rats except that incorporation of 14C radioactivity in the “metallothionein” fractions was less in kidney supernatant. The da& in Fig. 4 indicate that pretreatment of rats with CdClz increases levels of mstaUothionein-like protein whereas repeated exposure to mercury has no effect. In rats pretreated with CdClz both the 14C radioactivity and the absorbance at 254 nm increased in Sephadex fractions corresponding to the 10 066 mol. wt. region. Repeated exposure to mercury vapor produced table effect on % radioactivity and the absorbance at 254 nm in the IO ~~~ mol. wt. axon. Fu~er eh~a~~~zation of the newly synthesized protein in rat kidney after exposure to mercury vapor was achieved by comparing its heat stability
115
TABLE HI IN WV0 INCORPORATION OF [ tJ-14C] CYSTINE INTO LIVER AND KIDNEY OF CONTROL RATS AND RATS EXPOSED TO MERCURY VAPOR Rats were injected with 20 /Zi of [ 14C Jcystine (i.v.) 24 h prior to sacrifice. The experimental rata were exposed to stable metalliz mercury vapor for 7 daye before being injected with [ 14C Jcystine and were again exposed to mercury vapor fo., 2 h before sacrifice 24 h later. The results are Mean f SE. of three separate experiments. Three rats were used in each experiment. cpm/mg Protein
Liver Control Hg-Exposed Kidney Control Hg-Exposed --
Homogenate
Supernetant
816i 11 728 + 22
1228 * 42 1238 * 44
1057 f 35 1078 * 61
1247 * 51 1679 * 47
MfRC”RY VAWR
WOStD
CADMWM INJfCIfD
M FRACTION
40 WMBERS
Fig. 4. Sephadex G-76 fractionation of liver supernatants from control rats, rats expoeed to mercury vapor for 8 days and rats injected with CdCla for 2 days. [ i4C] Cystine (20 r Ci, i.v.) was injected 24 h before sacrifice. . - 1 - a, [i4C]cystine radioactivity; ----=*t absorbance at 254 nm.
116
Fii. 5. Get filtration control
on Sephadex
rats and rate exposed
with [ *%]cystine
G-75 columns of heated kidney supernatants
from
to metallic mercury vapor for 8 days. Rats were injected
(20 rCi, i.v.) 24 h before sacrifice. 105 000 g su~rnatant
was prepared
from kidney, heated at HP for 2 min and centrifuged at 10 000 g for 10 min. . - e- *, [ W]cyatine radioactivity; - - - - - -, absorbance 254 nm.
and isoelectric point with metallotbionein [ 15 ] . The data in Fig. 5 indicate the effect of heating the kidney supe~a~t from control and exposed animals. Heating is known to cause precipitation of high molecular weight proteins but to leave metallothionein unaffected [20]. Comparing the data in Fig. 5 with those in Fig. 3, it is apparent that heating the *4C-labeled supemataitt from kidneys of exposed animals results in loss of ~dioac~~~ specifically from the high molecular weight protein peak and complete recovery of r~~oactivity in the me~lothionein region.
0
IO
M
30
40
so
6’1
70
II0
1x3
FRACTION NUMBERS
Fig. 6. The profiles of WHg radioactivity (a - * - .) and absorbance at 254 nm (- - - - - -1 following isoelectric focusing of the 10 000 mol. wt. fraction from kidneys of rats repeatedly exposed to etemenlal mercury vapor. (For details, see MATERIALS AND METHODS.) The pii of the fraction is indicated by the continuous line.
117
A partial pu~fi~ation of the 10 000 mol. wt. proteins was ~~~rnp~ from rat kidney after repeated exposure to mercury vapor fo~owing the methods of Cherian /15]. The 10 000 mol. wt. fraction from Sephadex G-75 filtration was subjected to isoelectric focusing using ampholines, pH 3-6. Two protein peaks and corresponding mercury peaks were observed (Fig. 6), the isoelectric points being at pH 4.5 and pH 4.9. About 80% of the total radioactivity ap plied on the column can be recovered in the two main mercury peaks. These results are similar to thclise reported previously for isoelectric focusing of metallothionein induced in rat liver on pretreatment with cadmium salts [1!5] _ In a recent report, Nordberg et al. (211 have observed different isoelectric points (above pH 11) for mercury binding proteins in rabbit kidney after a~~ist~tion of mercuric chloride. DISCVSSION
The experimental evidence presented in this report indicates that repeated exposure of rats to elemental mercury vapor leads to the induction of metallothionein-like protein if not metallothionein itself. The size of induction is probably the kidney but confirmation would require in vitro experiments. The evidence may be briefly summarized as follows: (I) Repeated exposure of rats to mercury leads to a shift in the subcellular dis~bution of mercury in kidney tissue with most of the mercury being deposi~d in the soluble fraction. Further frac~ona~ons of the 105 OOOg ~pema~nt indicates that more mercury is bound to a 10000 mol. wt. fraction in rats having mtiltiple exposure as compared to animals reccPiving a single exposure to mercury vapor (Cherian, unpublished data). (2) Repeated exposure to mercury vapor results in an increased absorption at 254 nm in the 10 000 mol. wt. fraction from kidney supematant - a result similar to that obtained when metallothionein is induced by CdC12. (3) Repeated exposures to mercury vapor result in changes in the subcellular distribution of injected tracer doses of *09Cd, favoring its binding to the 10 000 mol. wt. fraction. (4) hollowing the approach of Piotro~ki et al. [3] the rate of in vivo ~co~oration of [UJ4C] cystine was taken as a measure of the biosyn~es~ of ‘~me~lo~ionein-like” proteins. Repeated exposure to mercury vapor enhanced the biosynthesis in kidney, but not in liver, (5) The similarity of the newly synthesized protein with metallothionein is also evident from its heat stability and its isoelectric points. The diffctrence in isoelectric points of proteins in the present study and that of NordMrg et al. [Zl] in rabbit kidney is not clear. However, it should be mentioned that pI more than 10 is usually associated with proteins like histones. (6) That the kidney is the probable site of synthesis is indicated ?,y the findings that the newly synthesized protein can be detected in kidney tissue but not in liver, These results confirm the findings of Sapota et al. 1221 on studies of rats repeatedly exposed to mercury vapor. These authors reported similar results 118
in pregnant rata but fetal levels of metahothionein were not increased by maternal exposure to mercury vapor. The effecb of exposure to mercury vapor are similar to those reported by Piotrowski et al. [3], in rats repeatedly dosed with mercuric chloride. This similarity ie not surprising since elemental mercury vapor is rapidly converted to ionic mercury in animal tissue and, as with HgCl*, accumulates preferentially ig the kidneys [ 181. The reason that exposure to mercury vapor does not lead to the induction of pro&An in the liver is probably due to the much lower levels of mercury in the liver, as is disc by ~o~~ki et al., [3] in expiring their similar with repeated doses of inorganic salts of mercury. find gnificance of these findings to human exposure to elemental mercury T vapor deserves 91brief comment. These experiments were made at high vapor ~oncent~~ons (about 4 mg Hg/m3). occupations exposures are at least an order of m itude lower than this. However, exposure times in our experiments were brief (2 h), and the animals were exposed for not more than 8 days. The induction of metallothionein in kidney tissue and the binding of mercury to this protein may be one explanation for the persistence of mercury in utile long after cessation of occupational exposure [23] and for the reports of human tolerance to mercury vapor in long-tea occupa~on~ exposure [ 241.
We wish to ~~k3udi assistance.
Allen and Ron Vander Mallie for excellent technical
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