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Brit. J. industr. Med., 1-969, 26, 139-143 Intracellular distribution of lead in the liver and kidney of the rat N. CASTELLINO AND S. ALOJ Institute of Industrial Medicine and institute of General Pathology, University of Naples, Naples, Italy Castellino, N. and Aloj, S. (1969). Brit. 7. industr. Med., 26,139-143. Intracellular distribution of lead In the liver and kidney of the rat.-The distribution of lead in intracellular fractions of liver and kidney has been studied in rats for nine days after intravenous injection of 100 /rg. of lead (M0Pb) per rat. The radioactivity and the protein nitrogen have been measured in the homogenates and in the nuclei, mitochondria, microsomes, and cell sap. Lead penetrates the cells quickly, being present in all fractions one hour after injection. During the first 72 hours the relative amounts of lead in the different fractions varied, which ruled out the possibility that all the differences could be explained by redistribution after homogenization. The dis tribution of the metal within the cell probably depends upon differences in the nature and the stability of bonding to intracellular structures. The binding of lead within the mitochondria seemed to be particularly stable, only 24-4% of the radioactivity being removed by washing with 0-25 m sucrose or 0-001 m ethylcnediaminetetra-acetate or 0 001 m diethylenetriaminopenta-acetate in 0-25 m sucrose. This finding is discussed in relation to haem biosynthesis within the mitochondrion. Previous work on the kinetics of distribution of lead in the rat indicated that the metal diffuses into the extravascular space and penetrates the cells, where it is probably fixed as an organic complex (Castellino and Aloj, 1964). Analysis of the data obtained in rats treated with a chelating agent, ethylene-diaminetetraacetltej confirms the above hypothesis, since different rates of removal of *"Pb from the tissues have been observed (Castellino and Aloj, 1965). probably due to inhibition of cytoplasmic and mitochondrial enzymes (Goldberg, Ashenbrucker, Cartwright, and Wintrobe, 1956; Rubino, 1961; Rimington, 1951; Eriksen, 1955; Dresel and Falk, 1956; Gajdos and Gajdos-Torok, 1958; Bernard, Gajdos,and Gajdos-Torok, 1958;Grinstein, Bannerman, and Moore, 1959), it was ofinterest to examine tbe distribution of lead inside the celts of liver and kidney homogenates after injection of 210Pb to rats. `'These results are in agreement with the findings of Bolanowska, Piotrowski, and Trojanowska (1964), who described the complex kinetics of lead in rats injected with a single dose of lead labelled with 210Pb in terms of three compartments with different exchange rates. Miani and Viterbo (1958) studied the distribution oflead in tissues ofthe dog by histo-autoradiographic techniques. They reported that the metal is located in various intracellular structures. Since one of the most important biological effects of lead is its7 interference with haem biosynthesis. Methods Animals Forty-six Wistar male rats weighing from 190 to 240 g. were used Tracer compounds moPb acetate, specific activity 50 mCi/mM, was obtained from the Radiochemical Centre, Amersham, Bucks. For injection this was diluted with 0-0001 N acetic acid in 0-9% Nad in water. Radioactivity determinations were carried out employing a well-type crystal scintillation 139 < N36919 a l:/< T> 140 N. Castellino and S. Aloj counter (Nuclear, Chicago) by estimating the gamma Results * emission of 21"Pb. Intracellular distribution of 210Pb in liver and kidney Treatment of rats Injections were given into the femoral vein under ether anaesthesia. Each rat was given 0-2 ml. of lead acetate solution, pH 4-7, containing 100 jtg. of lead. Radioactivity was 10* counts per minute (c.p.m.). The distribution of lead in the intracellular fractions of the liver and kidney was studied in six groups of five rate. The animals were killed, at intervals up to nine days after injection. An additional group of 16 rats, killed 24hours aftertreatment, was used only to prepare liver and kidney mitochondria. Analytical methods The liver and kidneys were removed, minced thoroughly, and washed several times in cold 0-25 m sucrose, thus eliminating most of the contaminating blood. Since it had been previously shown (Castellino and Aloj, 1964) that the concentration of lead per gram of blood accounted for only 1-23 and 0-03 % of the injected dose at one hour and nine days after injection respectively, perfusion of the organs was considered unnecessary. Separation of intracellular particles was accomplished by the method of Hogeboom, Schneider, and Pallade. (1948). Homogen ization was carried out on small amounts of tissue in a Potter-Elvehjem homogenizer with a Teflon pestle spinning at 700 r.p.m. Each portion was homogenized for 30 seconds. The homogenate was suspended in 0-25 m sucrose, 6 ml./g. fresh weight of tissue. Smears were observed under the phase contrast microscope to ensure that the cells had been broken. By centrifugation at 700 g for 10 minutes the nuclear fraction was isolated. Mitochondria were sedimented from this supernatant hy centrifuging at 21,000 g for 10 minutes. Finally, the microsomal fraction was isolated at 103,000 g for 60 minutes. The remaining supernatant will be called the soluble fraction. The nuclear fraction and mitochondria were washed twice with 0-25 m sucrose. Each fraction was weighed and the volumes of washing liquid were measured. The radioactivity and the protein nitrogen (Gomail, Bardawill, and David, 1949) were determined In the homogenates, in each intracellular fraction, and in the washing liquids. The amount of 210Pb was expressed in counts per minute (Table 1) while the values shown in the figures are corrected for nitrogen content by the formula: Table 1 shows the radioactivity of the homogenates and of each fraction at different times. Each result is the mean of five rats. Recoveries ranged from 100-2 to 121-0% in the liver, and from 106-5 to 117-5% in the kidneys. In. Figs 1 and 2, radioactivities are shown calculated from the formula in the Methods section, i.e,, radioactivities relative to the radio activity of the homogenate on a scale such that 100' indicates equal radioactivity/mg. N. At all times after injection all fractions contained lead. In the liver (Fig. 1) during the first 24 to72 hours we observed a decrease of the relative radioactivity in the nuclear fraction and an increase in the mito chondria, indicating different rates of lead removal from these intracellular structures. In contrast, no c.p.m./mg. N in fraction x 100 c.p.m./mg. N in the homogenate In order to study further the removal of the metal from the mitochondrial fraction, liver and kidney mitochondria from 16 rats injected 24 hours previously were prepared as above and divided into four equal parts, one of which was used as a control. The others were washed twice either with 0-2S m sucrose or with ethyienediamine tetra-acetate (E.D.T.A.) or diethylenetriaminopeiitaacetate (D.P.T.A.) both 0-001 m in 0-25 m sucrose. The radioactivity was determined in the mitochondria and in the washing liquids. In Table 2 the values are reported as percentages of radioactivity present in the unwashed control fraction. f ig . 1. Distribution of 2l<lPb in liver fractions relative to !I0Pb in the whole homogenate corrected for nitrogen content. S.E.M.S are shown.- f ig . 2. Distribution of 21*Pb in kidney fractions; as for Figure 1. DU P050312639 * Intracellular distribution of lead in the liver and kidney of the rat 141 TABLE 1 p oAmo u n t s o f 2I0Pb in Liv e r a n d in Kid n e y (Tis s u e a n d Su b f r a c t io n s ) a f t e r In j e c t io n o p 100 . . Le a d p e r 'Ra t : Va l u e s Ex p r e s s e d in Co u n t s p e r Min u t e x I O'1 (p e r c e n t o f In j e c t e d Do s e) Time (hours) Homogenate Nuclei 1 17-7 6 13-8 24 7-7 72 3-32 144 1-59 216 0-95 5-84 4-28 1-92 0-57 0-25 0-15 Washing liquid 4-72 3-92 1-79 0-55 0-23 0-12 Liver Mitochondria Washing liquid 1-23 1-14 0-98 0-62 0-30 019 0-24 0-22 0-20 0-13 0 05 004 Microsome 3-61 2-36 1-98 0-99 0-48 0-29 Soluble fraction 5-76 4-24 2-08 0-87 0-32 0-21 Recovery % 121-0 116-3 114-5 112-3 100*2 105-2 Time (hours) Homogenate Nuclei t 201 6 18-3 24 . 12-7 72 5-75 144 2-81 216 1-64 5-16 4-18 3-66 1-64 0-74 0-43 Kidney Washing Mitochondria Washing liquid liquid 5-14 4-12 3-70 1-52 0-76 0-45 2-41 3-42 3-40 1-51 0-68 0-37 0-48 0-66 0-62 0-31 0-16 0-07 Microsome 3-47 2-88 1-22 0-47 0-18 Oil Soluble fraction 6-12 4-88 2-31 1-14 0-47 034 Recovery % 1130 109-7 117-5 114-6 106-5 107-3 important variations occurred in the relative radio activities in the rnicrosomes and in the soluble fractions. At later times the relative radioactivities in all intracellular fractions became almost constant, i.e., all lost radioactivity at the same fractional rate as the liver as a whole, indicating the setting up of an equilibrium state. In the kidney (Fig. 2), during the first 24 hours after injection, we observed increases of the relative radioactivities in the nuclear and the mitochondrial fractions, a decrease in the rnicrosomes, and little change in the soluble fraction. After 24 hours the distribution of lead inside the kidney cells reached an equilibrium state, as indicated by relative radio activities which remained almost constant up to nine days. Removal of 210Pb from mitochondria As reported under Methods, during the isolation of fractions the nuclei and mitochondria were washed twice with 0-25 m sucrose. The washings removed about 50% and 20% of the radioactivity from the nuclear and mitochondrial- fractions respectively. . Mitochondria from the liver and kidneys of 16 rats injected with 210Pb were washed with sucrose, E.D.T.A., and D.T.P.A. solutions (see under Methods). Table 2 shows that 80-0 to 87-6% and 80-2 to 86-2% of radioactivity remained after two washes in liver and in kidney mitochondria respec tively. The high proportion of radioactivity which remained bound to the mitochondria after repeated TABLE 2 Pe r c e n t a g e o f ai0Pb in t h e Mit o c h o n d r ia l Fr a c t io n s a f t e r Wa s h in g a n d in t h e Wa s h in g Me d ia . Washing medium Sample Liver Kidney %% Sucrose, 0-25 M .. Mitochondria First wash Second wash 82-4 20-3 4-i 80-2 i8-4 2-1 E.D.T.A. (mM) in sucrose. Mitochondria 0-25 M First wash Second wash 87-6 15-3 0-0 82-0 12-4 2-1 D.T.P.A. (mM) in sucrose. 0-25 M Mitochondria First wash Second wash 80-0 14-5 0-0 86-2 1S-1 0-0 Figures refer to 1 mg. of mitochondria or 1 ml. of washing medium as percentages of the radioactivity in the unwashed mitrochondrial fraction. washing shows that the binding was remarkably strong. Discussion Lead poisoning is characterized by a disorder in 'haemoglobin synthesis, as shown by increases in haem levels and in the urinary excretion of some ' " 4, ASi, ir -i t- 1 i i DU P 050312640 * f r ! \ : i i;< l- i. ~V * .TV- ; W- 142 N. Castellino and S. Aloj haem precursors (Schmid, Hanson, and Schwartz, 1952; Tishkoff, Granville, Rosen, and Dameshek, 1958; Sano, 1958; Danieli, Gajdos-T6rok, and Gajdos, 1961; Pecora, Fati, Mole, Balletta, and Daniele, 1963; Crepet and Rubino, 1956; Rubino, 1961). It is well established that heavy metals can com bine with a large variety of organic molecules by interaction with ligands such as sulphydryl, carboxyl, amino, imidazolyl or phosphoryl groups (Passow, Rothstein, and Clarkson, 1961). We think that, because of these interactions, lead may behave as an inhibitor of various enzymes involved in haem biosynthesis. It is therefore interesting that recent studies have established that some important reactions involved in haem biosynthesis take place in the mitochondria-decarboxylation and oxida tion of coproporphyrin III to protoporphyrin IX; incorporation of ferrous iron into protoporphyrin IX with formation of haem; and, probably, con densation of S-aminolaevulinic acid to porphobilin ogen (Dresdel, 1955; Sano, 1958; Minakami, Yoneyama, and Yioshikawa, 1958; Eriksen, 1960; Lascelles, 1964; Rlet, 1964; Policard, 1964). The findings reported in this paper show that, within a short time after injection of lead, the metal is incorporated in all intracellular fractions obtained by differential centrifugation of liver and kidney homogenates. Some variations in the relative amounts of 210Pb in the intracellular fractions were observed up to 72 hours after injection, indicating different rates of removal of lead from each fraction. These variations rule out any major redistribution of lead in vitro after homogenization, and suggest that the differ ences in concentration inside the cell probably depend upon the nature and the stability of lead binding to intracellular structures. In both liver and kidneys the 210Pb present in the mitochondria decreased more slowly than in the other fractions and in the whole homogenate. From 72 hours to nine days from injection the relative amounts of 210Pb in the fractions remained constant, suggesting that'a steady state had been reached. The slowness with which this equilibriumis reached may depend either upon a strict compartmentation between the different phases in which the lead is distributed inside the cells or upon a slow transformation of the molecular species of the metal in the celL The failure to remove 210Pb from mitochondria by washing suggests that the binding of lead within the mitochondrion is very strong. This seems to be in agreement with the hypothesis of direct interference by lead with some steps in haem biosynthesis (mainly occurring in the mitochondria), probably due to an enzymatic block of S-aminoIaevuIinatedehydratase and haem synthetase. References * Bernard, H., Gajdos, A., and Gajdos-Tdrbk, M. (1958). Biognise de I'himogiobme par le sang pdriphferique da lapin. Action du plomb du florare de sodium et de 1'oxyle de carbone. Expos arut. Biochlm. mid., 20, 41*54, Bolanowska, W., Piotrowski, J,, and Trojanowska, B. (1964). The kinetics of distribution and excretion of lead (Pb") in rats. In XlVth int. Congr, accup. Wth, Madrid 1963* Vol. 2, pp. 420-422. tExc. tned. Snt. Congr. Ser., No. 62J. Castellino, N., and Aloj, S. (1964). Kinetics of the distribution and excretion of lead in the rat. Brit. J. industry Med., 21, 308*314, ------ ------ (1965). Effects of calcium sodium ethylenediamiite-tetra- acetate on the kinetics of distribution and excretion of lead in the rat. Ibid.. 22, 172*180. ' Crepet, M., and Rubino, G. F. (1956). Recenti acquisizioni sol satunusmo. Parte I: Aspetti einatolcgiri deHa intossicaziane. In XXI Nat. Congr. Med. Lavoro. pp. 5-63. Danieli, G. Gajdos-Tdrdk, M., and Gajdos, A. (1961). Un nouveau test biologiqoe du saturnisme: augmentation do taux urinaire et plasmatique de Vadde delta-aminolfevuirmqoe. Path, et Biol., 9, 1481-1486. Dresel, E. I. B. (1955). The rdle of some porphyrins and porphyrinprecursors in tbe biosynthesis of haem. In Ciba Foundation Symposium on Porphyrin Biosynthesis and Metabolism. pp. 72-85. * Churchill, London. ------ , and Falk, 3. E. (1956). Studies on the biosynthesis of blood pigments. 3. Haem and porphyrin, formation from 3-amiaolaevulmic add and from porphobilinogen in baemolysed chicken erythrocytes. Biochem. J.t 63, 80-87. Eriksen, L. (1955). Lead intoxication: 1 the effect of lead o zl the in vitro biosynthesis of haeme and free erythrocyte porphyrins. ScandJ. dirt. Lab. Invest, 7,80-85. ------ (I960). The rdle of protoporphyrin, iron and globin in haemo globin formation, and the possible fate of excess in formed protoporphyrin, Les maladies du mStaboEsme des porphyrines. 2 Congr. int. Biol. Saclay, pp. 217-234. Presses Universitaires, France, Gajdos, A., and Gajdos-Tdrdk, M. (1958). Anmie bypochrome, hypersidirimique expdrimcntale par intoxication saturnine, fluorge on oxycarbonfie du lapin. M&amsme pathogfinique. Sang., 29,444-460. Goldberg, A., Ashenbrucker, H., Cartwright, G. E., and Wintrobe, M. M. (1956). Studies on the biosynthesis of heme in vitro by avian erythrocytes. Blood, It, 821-833. Gornall, A. G., Bardavvill, C. J., and David* M. M. (1949). Deter mination of serum proteins by means of the biuret reaction. 7. bioh atom., 177, 751-766. Grinstein, M., Bannerman, R. M., and Moore, C, V. (1959). The utilization of protoporphyrin 9 in heme synthesis. Blood, 14, 476-485. Hogeboom, G. H., Schneider, W. C., and Ballade, G. E. (1948). Cytochemical studies of mammalian tissues. 1 Isolation of Intact mitochondria from, rat liver; some biochemical properties of mitochondria and subrmcroscopic particulate material. X blot. Ghent., 172,619-635. Lascelles, 3. (1964). Tetrapyrrole Biosynthesis and its Regulation. Benjamin, New York. Miani, N., and Viterbo, B. (1958). Studio istoautoradiografico sulla localizzazione del piombo (RaD) in van organs di cane. Z. Zellforsch., 49,188-208. Minakami, S., Yoneyama, Y., and Yoshifcawa, H. (1953). On the biosynthosis of heme and hemeprotems in fiver cell. Biodmn. blophys. Acta (.Antst.), 28, 447-449. Passow, H., Rothstein, A., and Clarkson, T. W. (1961). The general pharmacology of the heavy metals. Pharmacol. Rev., 13, 185-224. Pecora, L. Fati, S., Mote, R., Balletta and Daniele, E. (1963). 11 comportamento del metabolismo porfrrinico nel satumlsmo con particolare riguardo all*ALA e al PBG. Folia med., {Napoli), 46, 107-124. Pilet, P. E. (1964). La Cellule. Masson, Paris. Policard, A. (1964). Cellules Vivantes et Population Cetlutaires. Structure et Fonctions. Masson, Paris. Rimington, C. (1951). Preliminary investigations for a 3tody ofenergy utilized by the surviving fowl erythrocyte in haem synthesis. In Ciba Foundation Conference on p Isotopes in Biochemistry, ed. G. E. W. Wolsienholme, pp. 86-89. ChurddH, London. Rubino, G. F. (1961). Tbe rdle of lead jn porphyrin metabolism. In Patmtin. Med., Symposium on the Normal and Pathological Metabolism ofPorphyrin, pp. 40-44. Sano, S. (1958). The effect of mithocondria on porphyrin and haeme biosynthesis in red blood cells. Acta haemat. jap., 21* 337-349. (Suppl. 2\ DUP050312641 Intracellular distribution of lead in the It ver and kidney of the rat 143 Schmtd, R., Hanson. B., and Schwartz, S. (1952). Experimental porphyria. I: Isolation of uroporphyrin I from bone marrow of lead poisoned rabbits. Proc. Soc. exp. Biol. 79, 459-462. Tishkoff, G. H., Granville, N. B., Rosen. R.. and Damcshek, W. (1958). Excretion of delta-aminolevulinic acid in lead intoxication. Acta haemat. {Basel), 19, 321-326. Received for publication May 14, 1968. ' *. The January (1969) Issue The occupation of trawl fishing and the medical aid available to the Grimsby deep sea fisherman S. R. W. Mo o r e The mortality and morbidity of deep sea fishermen sailing from Grimsby in one year S. R. W. Mo o r e Absence from work and physical fitness Vic t o r Lin d e n Sleep and wakefulness in a group of shift workers G. S. 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