Int. J. PeptideProtein Res. 10, 1917, 190-196 Published by Munksgaard, Copenhagen, Denmark N o part may be reproduced by any process without written permission from the author(s)

I N V E S T I G A T I O N O N T H E R E A C T I O N O F M O L Y B D E N U M WITH COLLAGEN IN VIVO BEDlhCH BfBR, ZDENEK DEYL, JAROSLAV LENER AND MILAN ADAM

Isotopova laboratof Biologickich dstav; a Fyziologickj ustav ?eskoslovenskh akademie vzd, Institut hygieny a epidemiologie, and Vizkumn); dstav chorob revmatickich, Prague, Czechoslovakia

Received 13 September 1976, accepted for publication 4 March 1977 Male Wistar rats were treated with Mo in a single dose or over a period o f 84 days. The course of molybdenum excretion after a single dose was expressed mathematically. I t was shown that various tissues produced different excretion curves and a high retention was observed only in skin. In long-term treatment it was possible to ascribe this retention to direct binding of molybdenum to collagen. In contrast to some other heavy metals molybdenum causes a decrease o f collagen stability. The mechanism of this effect is discussed.

It has been previously shown that heavy metals, mainly in the chelate form, react with collagen in vivo (Adam et al,, 1964, 1968a, 1970; Ryback, 1968; Eberl, 1971). The evidence for this interaction was obtained by electron microscopy and, particularly in the case of gold, also by direct analytical estimation (Adam & Kdm, 1968; Deyl et al., 1970). It has been shown that heavy metals are susceptible both to monotopical and bitopical binding, and in a limited number of interactions along the collagen fiber they form artificial cross-links. These newly formed cross-links are of considerable importance as far as the mechanochemical properties and solubility of the fiber is concerned. Metals administered in the previous experiments were in the cationic form or in the form of electropositive complexes. The object of these studies is mainly an attempt to elucidate the mode of action of gold complexes in the treatment of rheumatoid arthritis by gold therapy. At present no satisfactory data are available on the interaction of electronegative complexes 190

or anions of heavy metals with collagen in vivo., The reason why it appears desirable to elucidate this type of interaction is based mainly on the accwnulatory properties of connective tissue with special reference to trace elements an$ environmental hazards. Heavy metals, both in the cationic or anionic form, are the constituents of many pesticides or herbicides, engine exhausts and numerous other air or water pollutants. More specifically, the present experimental work is directed towards molybdenum and its anticariogenic effects. MATERIAL AND METHODS Male Wistar rats maintained on Larsen chow containing less than 0.3 p.p.m. of natural molybdenum and tap water were used. Single dose treatment with molybdenum

Retention and excretion of molybdenum wasc assayed in 2-month-old animals which were injected subcutaneously with a single dose of ammonium molybdate labeled with 99M0. Doses

INVESTIGATION ON THE REACTION OF MOLYBDENUM WITH COLLAGEN IN VZVO

administered were 25, 40, 70, 200, 400,800, 1700, 3000 ng 99 Mo per g ng/body weight and represented an activity of 50-lOOpCi per animal. Labeled ammonium molybdate was purchased from Radiochemical Centre, - h e r s h a m (Great Britain). Rats were killed by decapitation after2,4,6,10, 13.5, and 14 days. Organs investigated were removed and weighed, and the radioactivity was counted after isotope equilibrium between *Mo and 9 9 m T ~was reached, using the Gamma Automatic System Nuclear Chicago, Model 4224.

(NazMo04) were then mineralized with a mixture of HNOB and HC104 (3: 1) and taken nearly to dryness. The mineral substances were transferred with 40 ml of 1 N HCl into a separatingfunnel and 1 ml of 10% thiocyanate solution and 0.2 g of solid ascorbic acid were added and shaken. 2ml of a 10% solution of SnClz and 5 ml of methyl-isobutyl ketone were added to the reaction mixture, shaken for 2min (Kim et ~ 1 . . 1974) and the extract was assayed in the Gamma Automatic System Nuclear Chicago Model 4224.

Long-term treatment with molybdenum

Contraction-relaxation behaviour of rat-tail tendon collagen under a given load (50mg) was ascertained in 2 M KI (Chvapil & Zahradnik 1960). The original length of rat-tail tendons was 5 cm. Controls were of the same age as the investigated animals at the end of the experiment. For the electron microscopic studies the tail tendons, after 12 weeks treatment, were cut with a freezing microtome (5 pm), suspended in water without further treatment and picked up on membranes on microscope grids. Electron microscope studies were carried out with a Tesla-BS 613 electron microscope at 80 kV.

Contraction -relaxation

Experimental animals 21 days old were treated with sodium molybdenate p.a. in redistilled water over a period of 84 days. Molybdenum concentrations in water were 500, 800 and 1000p.p.m. Mo. Water consumption was estimated daily and the total uptake of molybdenum was established (6.97, 10.78, and 13.41 mgMo/ per g body weight). Collagen preparation for activation analysis 1

For the analytical estimation of molybdenum bound to collagen, skin samples were freed from subcutaneous tissue or fat and then extracted with 0.45 M NaC1-phosphate buffer, as published elsewhere (Adam etal., 19686). Activation analysis

Neutral salt-soluble collagen (0.5 g) from rats treated with medium and high doses of moly-bdenum and dried at 110' was irradiated with thermal neutrons over a period of 40h at a flux of neutrons. Samples with 1 mg of carrier

content was estimated in kidney, liver, testes and cross-striated muscle according to Stegemann (1958). Calculation of data and fitting of experimental values to mathematical formulae were done on a Hewlett Packard Computer Type 2116 C.

Hydroxyproline

TABLE 1 Content of molybdenum in organs of eight rats after a single dose of labeled ammonium molybdate (dose = 800 ngMolg b. wt.). Means i standard error

Time (days)

Kidneys ngMo/g b.wt.

Testes ngMo/g b.wt.

2 4 6 10 13.5 14

1399 i 106 1496 i 70 1576i 74 1407 f 106 446i 87

45 i 8 37f2 22 i 2 11.6 i 0.6 6.1 i 0.8

-

-

Crossstriated muscle ngMo/g b.wt.

Skin ngMo/g b.wt.

9.8 f 0.7 8.0 f 1.1 5.0 i 0.4 2.9 i 0.3

76 5 i 7.0 58 8 i 3.8 39.3 i 3.8 31.1 i 3.2

-

22.0 i 2.1

-

Liver ngMo/g b.wt.

508 i 22 292 f 17 235 i 17 1OOi 8 57i 4

-

191

B. BfBR, Z. DEYL, J . LENER and M. ADAM

RESULTS AND DISCUSSION Single-dose treatment of m o l y b d e n u m

The excretion of a single dose of molybdenum (800 n d g body weight) is illustrated in Table 1. With the other doses (see Material and Methods) the relations are analogous to the one described in Table 1. Compared with other organs, kidneys are specific in maintaining a constant level of molybdenum during the first 10 days after administration. About 14 days after administration the molybdenum content in kidneys starts significantly decreasing. In the liver, crossstriated muscle, and testes a continuous decrease in molybdenum concentration occurs, which can be characterized by a simple excretion exponential. This indicates that no retention of molybdenum occurs during a 2-week period. To avoid presenting a large number of data obtained with the various doses, mathematical treatment appeared desirable. Generally, excretion of molybdenum follows an exponential curve in all organs except skin, in which an additional constant, k, had to be ascertained to make the equation fit the experimental data. Excretion of molybdenum can be given thus:

where

Y

=

D

=

h t k, 1

=

= =

a, b, n

the equation has been proved (in days). = dimensionless constants.

Experimeptal data relevant to the above equation are summarized in Table 2 and permit comparison of the properties of the tissue investigated. The most characteristic value, which expresses the excretion rate, is A, inversely proportional to the excretional biological halflife (A = 0.6931/~days). As can be seen from, Table 2, experimental values for different tissues are not very different. The constant II* represents extrapolated initial concentration necessary to achieve a dose equal unity (D = 1 ng Mo/g b.w.). The constant n characterizes the proportionality between the concentration and administered dose. With the exception of skin,' the calculated values valid for different tissues, are also very close. From the viewpoint of retention the most important constant is b , which represents the retention of molybdenum in a particular tissue or organ within the investigated period of time. Out of the investigated tissues only skin exhibited a b value different from zero thus proving a strong capability of Mo retention. The absolute value of this constant is strictly limited by the Yt% = D" (a-At + b) time period investigated, and outside these limits conclusions would be hazardous. This, however, does not decrease the importance of molybdenum concentration in ng the finding that skin differs substantially in its Mo/g wet tissue. behaviour towards Mo in comparison with all administered dose of molybdenum other tissues investigated. in ng Mo/g body weight. These data are in good agreement with the excretion rate constant in days-' excretion and retention of molybdenum as time after administration in days. followed in the whole rat (Bfbr & Lener, time limits for which the validity of 1973). TABLE 2

Experimental values for equation [ I ) Organltissue

Validity (time interval in days) I + k days

n

a

retention constant b

h

7

(day-')

days

Kidney Liver Testes Cross-striated muscle

2-10 2-13.5 4-13.5 2-10.0

0.863 0.804 0.791 0.658

4.70 3.44 0.4 04 0.156

0 0 0 0

0 0.194 0.192 0.160

0 3.51 3.61 4.35

Skin

2-14

0.360

1.420

0.273

0.280

2.47

192

INVESTIGATION ON THE REACTION OF MOLYBDENUM WITH COLLAGEN IN VIVO

in electron micrographs. A typical example is presented in Fig. 2. The 70-nm As evidenced in Fig. 1 contraction of (Scm periodic unit contains a t least eight dark crosslong) rat-tail tendon collagen (RTTC) takes striations. The relative positions of individual place in seconds and more easily in samples TABLE 3 from animals treated with molybdenum than in controls. There was a significant difference in Collagen concentration in some organs of controls and experimental animals the degree of contraction after the doses 13.41 and 10.78mgMo/g body weight. With respect pg Hypro Significance to the lowest dose administered (6.97 mg Mo/g Organ Intake of Mo, mgMo/100g b.wt. l00mg dry body weight) there was no statistically signifi(84 days) weight cant difference in the degree of contraction, but a significant difference appeared in the 0 113 i 12 relaxation phase of the curve. Because an in- Liver 337 i 40 +++ 697 creased cross-linking of collagen structure 353 + 30 +++ 1048 results always in a higher contraction and pro+++ 252 i 28 1341 longed relaxation, the data summarized in Fig. 1 indicate that Rl’TC ‘Tom molybdate- Kidneys 0 211 f 1 1 treated rats was less cross-linked. The blockade +++ 396 i 35 697 of cross-linking is roughly proportional to the +++ 1048 393 + 28 amount of administered molybdate. Changes 1341 333 i 21 +++ observed with molybdenum-treated RTTC are 0 557 i 56 just the opposite, compared with controls to cross697 447 + 11 + those observed after the administration of other striated 1048 532 i 38 + heavy metal chelates like gold, lead, copper, muscle 1341 436 i 52 + nickel, bismuth, etc. (Adam et al., 1970). Assay of the collagen concentration in selected 0 472 i 35 organs showed that in all studied organs except Testes 697 782 i 43 +++ muscle the concentration of hydroxyproline 1048 633 i 37 ++ was elevated after molybdenum treatment 1341 637 i 29 ++ (Table 3). The amount of molybdenum bound in the collagen structure after long-term treat- N.S. + ment must be quite high since a distinct cross- P < 0.01 ++ striation pattern can be observed in rat-tail P