ENVIRONMENTAL
RESEARCH
Cadmium
29-38
(1975)
Accumulation in Mouse Organs During Sequential Injections of Cadmium-109’
JUNKO School
10,
MATSUBARA-KHAN'AND qf Health
Sciences.
Faculty
KAZUHIKO of’ Medicine
the
MACHIDA
Uni\,rrsity
qf Tokyo.
Tokyo. Japnr1 Received
August
27. 1974
to demonstrate the accumulation Sequential injections of ‘OYCd on mice were performed patterns of cadmium in various organs in mammals at tracer level. During the experimental period of 130 days, slow and upward accumulation of the tracer continued in most organs except gonads in which the equilibrium was attained. These linear increments showed slightly downward projection as sigmoid, which suggested the fitness to the two-comamong difpartment-model. More than a thousandfold difference of ‘O”Cd concentrations ferent organs was observed, and these striking differences of tissue levels manifested the strong tissue specificity of cadmium distribution. Sexual variation was also demonstrated; in blood, stomach, liver, spleen and kidneys the tracer concentrations in females were from I. I to 1.5 times those of the males, while in the skeletons males kept a 1.5-fold higher concentration of the tracer.
INTRODUCTION
Virtual absence of cadmium turnover in the plasma of rat was reported by Cotzias (196 1). Schroeder ( 1961) noticed that cadmium behaves differently from other ubiquitous metals in mammals and is concentrated in the kidney against the zinc-cadmium concentration gradient. The unique character of cadmium turnover was further traced recently by Matsubara-Khan (1974a, 1974b) in the process of compartmental analysis of the data from the tracer experiments aimed at obtaining biological half lives of inorganic cadmium, mercury, zinc, chromium and selenium in various organs of mice. The quantitative behaviour of cadmium in animal organs can be characterized by the slow turnover as the data gave unusually small rate-constants and/or gave extremely long biological half-lives in comparison to other metals; thus in some organs data fitted the two-compartment model well, which suggested the presence of relatively compact metabolic barrier among the organs. Various reports on cadmium retention in mammalian tissues were mostly concerned about single administration experiments while data on cadmium accumulation during a sequential administration are scant. Decker ( 1958) observed linear increase of cadmium concentration in rat kidney and liver after continuous oral administration for 6 and 12 months. Accumulation patterns of cadmium in rat liver and kidney were also reported by Bonnell et al. (1960) and that of mouse whole body in the review of Nordberg (1972).
r All communications
regarding
this paper
may be sent to J. Matsubara-Khan 29
Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
30
MATSUBARA-KHAN
AND
MACHIDA
The present studies aimed at demonstrating the patterns of cadmium accumulation and the difference of concentration levels in various organs of mice as a result of prolonged sequential injections of radioactive cadmium in order to confirm their fitness to the compartment models for the sequential uptake experiments. MATHEMATICAL
MODELS
In the previous paper Matsubara-Khan (1974a, 1974b) the fitness of compartment models for the single injection systems at individual and organ levels of observations has been discussed, while this time discussions are concentrated on the application of compartment models for the sequential administration systems. The theoretical models are shown in Fig. 1 and underlying hypothesis and experimental situations were similar to the previous paper by the author, except that the tracer was sequentially injected twice a week up to the time of the autopsies. One-Compartment
Model for the Sequential
Irgection
Systems
The dynamics of the amounts of the tracer in an organ, which is regarded as one compartment q(t), is to be regulated by the rate of tracer uptake to the organ II, and the rate of tracer elimination which is the product of q(t) and a rate constant k. This relation is expressed as follows: -&W = M - k . q(t). dt
(1)
When u is constant, the integration of Eq. (I) between t = 0 and t = t with initial condition q(0) = 0 gives a solution, q(t) = f ( 1 - e-/i’t). If data are obtained as the relative substituted as follows:
concentration
(2) of the tracer, Eq. (2) can be
y(t) = z (1 - eCh”f)
B
t
up ’
9s (*I .[YW]
(3)
1
k’
I q(t)
/ .
[y(t)]
k I
4
FIG. I. The theoretical models for the turnover of the metal in the studies of sequential injections systems. For explanation see text. (A) One-compartment model in the sequential administration system. (B) Two-compartment model in the sequential administration system.
Cd
UPTAKE
IN
MOUSE
ORGANS
31
where y(t) is the concentration of tracer in organ, e.g., cpmlg wet tissue, provided that the observations are carried out using the animals in a steady state without noticeable growth. T\iso-Compar.tment Model.for the If the tracer reaches the organ ment, in a manner similar to the postulate the following equations second compartments,
Sequential Injection Systems under observation via a preceding compartone explained in the previous paper, we can for the amounts of tracer of the first and the
-dq’(t) = 11’ - A’ . q’(f) dt
-ddf) = k’ * q’(t) - k . q(t) dt where q’(t), ~1’ and I;’ are the amount of tracer, rate of uptake and rate constant of the first compartment and q(t) and li are those for the second ones, respectively. The solution of the above equations is given as follows with an initial condition that q’(O) = 0, q(0) = 0 at t = 0, 1 -k
k-k’
e -k-‘l
In terms of y(t) instead of q(t), we can get an equation similar to Eq. (6) where 11’ should be multiplied by the weight factor of both compartments, i.e.. W/W’. EXPERIMENTAL
Sixty male and 60 female ICR mice 8 weeks-of-age were utilized in the present study. With intervals of 3.5 days namely twice a week, carrier free ‘OsCd Cl, 6.5 &i/kg body weight was sequentially injected intraperitoneally to all animals until they were autopsied. At each time of injections the tracer was injected to each mouse with strict proportion to his body weight. Mice were kept at room temperature around 15°C and fed with ordinary mouse feed. Autopsies were carried out 11 times during the 130 days of experimental period. Each time five males and five females were sacrificed. Immediately afterwards, ten organs from each individual were taken, weighed and radioassayed with Aloka DC-6 well-type scintillation counter. RESULTS
Patterns of Cadmium
Accumulation
in Mouse Orguru
The concentrations of losCd, cpm/g wet tissue in each organ were plotted against time in days as shown in Fig. 2 (A-H). In each figure the open circles show the averages of data obtained from five females and closed circles show the average values from five males. From the results of single injection experiments described in the previous study ( 1974a), it was expected that the turnover speed of cadmium in kidneys and
Xl,O'CPM/y XlO'CPM/y ,%. A. KIDNEY 150
xlg+cPM /g 8. LlVER
loo100 . _.I
5050 & IIII, x1o*cPM/g g 1625 3544
I,,
5665
77
, , II,,, I I I I I 91 103DAysM) XlO+CPM/g 3 1625 35 44 56 $5 77 9'1 103mYS130
%
D. at%%
x20
III,, 916 25 3544
I,, 5665
77
, , I 91 103mys 130
1 I 9 169
I'll I1 b r 35 44 56 65 77 91 103D,&30
0
E. SPLEEN
91625 XlO+cPM&
3544
5665
77 $1 103 DAYS13 916 253544 0 X104CPMkJ
G. BONE 1.Q
0
.'o
0
l
5665
i7
91 103D4ys130 0
H. LUNG
8
0
6
0
/
45
916 25 35 44 56 65 77
91 103DAYS130
9 16 25 35 44 56 65 77
91 103DAys130
FIG. 2(A-H). Patterns of cadmium retention in various organs during the sequential injections of “Td to the mouse. Open circles show the averages of data obtained from five females, and closed circles show the average values from five males. Solid line shows the trend obtained from the averages of the observed data, while dotted line in each graph of A-D shows the theoretical curve of Eq. (6) calculated from the previously obtained rate constants in the single injection experiments and presently extrapolated equilibrium value. For detailed explanation see Discussion. Calculated equations
are as follows.
y = $
B 1 - __ B-C
e-et. ~ ‘B-C
C
where in graph A, kidney, were param>’ eters A = 1.24, B = 0.001. C = 0.30; in graph B, liver, A = 0.60, B = 0.01, C = 0.57; in graph C, salivary glands, A = 0.026. B = 0.002, C = 0.70: and in graph D, female gonad, A = 0.50. B=O.OlS, C= 0.17 and malegonad,A = 0.26, B =0.019, C=O.O52. (
32
e?r
Cd
UPTAKE
IN
MOUSE
ORGANS
33
salivary glands must be very slow because they gave extremely small rate constants. The present results showed that the turnover speed is slower not only in the above mentioned organs but also in most of the organs with the exception of gonads. In more than 4-months observation, slow upward accumulation of radiocadmium continued, only the data from gonads seemed to have attained steady state. These linear increments showed slightly downward projection. This pattern is quite different from the uptake pattern of other metals which fit the Eq. (3) of the one-compartment model having upward projection, hyperbolic curve. The accumulation pattern of ‘O”Cd in kidneys was sigmoidal (see Fig. 2A). This was quite in accord with our expectation, since our previous results of single injection data from kidneys had fitted the two compartment model. Tissue Concentration
Levels of “‘Cd
In Table 1 are shown the mean concentrations of ‘O”Cd from five males (upper) and from five females (below) in ten organs during the experimental period. Remarkable differences were observed in cadmium concentrations of various organs. On 130th day, the end of the experimental period, the relative concentration in kidneys was about 150; liver, 50; spleen, 30; uterus and ovary, 25; testis and seminal vesicles, 10; stomach or lung, 10; salivary glands or prostate glands, 3; skeletons, 1.5; pelt, 1.3: muscle, 0.6; brain, 0.1; and blood of males 0. 122 and blood of females l.6,2 respectively. These clear differences showed strong tissue specificity of cadmium distribution. Sexual Variation As the tracer was injected strictly proportional to the body weights of experimental animals, the data presented in Table 1 provide the comparison of the concentrations in males and females. It was noticed that females had generally higher concentrations. In blood, stomach, liver, spleen and kidney, the tracer concentrations in females was roughly 1. l- 1.5 times those of the males, while in the skeletons it was vice versa. DISCUSSION
More than a thousandfold difference of tracer concentrations among different organs of the mouse was observed. Highest concentration was observed in kidney with relative concentration of 150, while lowest one was in brain at 0.1. In these experiments, data had usually 20-40s of coefficients of variance. Standard errors of mean values were shown in Table 1, the data however manifested consistently clear differences of the tissue levels with repeated observations of eleven series of autopsies. This should be noticed as unique character of cadmium distribution which is different from zinc. Stable cadmium concentrations surveyed by atomic absorption spectroscopy in a separate study showed only hundredfold difference among the various ‘Nofr: Cadmium-109 in blood was assayedonly on the 130th day’s observation. Male average of five animals was 1924 cpm/g with standard error 572 and female averageof six animals was 23875 cPm/g with standard error 437 I. Though this difference was noteworthy, we could not confirm such noticeable sexual difference in a later study of atomic absorption spectroscopy; a confirmatory tracer experiment for this is not yet finished.
16
25
35
44
56
65
77
91
103
130
2
3
4
5
6
7
8
9
10
11
M F M F M F M F M F M F M F M F M F M F M F
Sex
’ 1a each column, standard errors.
9
Day
1
No.
Autopsy
c c * + t_ * 2 ? + i * i + + F + C f t &
upper
6.12-c 8.59~ 12.0 12.1 21.1 19.9 21.5 23.8 38.6 49.6 46.8 63.0 51.6 52.7 71.1 80.7 113.0 112.0 132.0 138.0 155.0 164.0
Kidney
values
0.64 1.15 1.2 1.2 1.7 1.2 1.9 1.8 2.2 11.9 4.1 3.8 6.5 5.4 9.9 7.1 8.0 9.0 12.0 15.0 15.0 14.0
show
4.74 6.63 7.61 8.96 9.45 11.3 12.4 14.5 14.3 15.3 15.8 21.1 12.7 14.8 20.8 32.1 22.5 36.1 32.4 38.1 36.0 51.0
the average
TABLE
tracer
t 0.25 i 0.32 k 0.14 1.13 k 0.71 k 0.63 i 1.22 -+ 0.88 k 1.17 k2.1 k 1.7 ~3.6 k 1.7 kO.8 i- 2.3 k 3.2 ~3.8 24.8 k5.8 -c3.3 k6.2 i3.3
Spleen
k t t_ k k 2 k k t -t k ?I -c f t k k t -t _f i 2
concentrations
0.491 1.29 0.810 2.13 1.46 2.04 1.95 3.38 2.26 5.53 2.61 5.02 4.55 6.85 3.43 6.83 3.35 6.45 6.93 9.82 7.40 12.3
Stomach
0.235 0.298 1.04 0.765 0.784 0.594 1.61 0.748 1.93 1.13 2.72 2.50 4.83 1.86 4.40 4.35 2.72 3.30 4.30 6.99 5.43 8.41
of 5 males
0.107 0.16 0.068 0.20 0.25 0.11 0.30 0.54 0.35 0.78 0.25 0.82 0.37 0.59 0.61 1.78 0.55 1.11 0.70 2.56 0.23 11.1
0.017 0.051 0.40 0.329 0.163 0.124 0.39 0.075 0.72 0.12 0.94 0.88 1.52 0.12 1.19 1.57 0.27 0.89 0.52 3.07 1.70 1.97 with
k + _f f_ k t 2 t + + t ik t -c 2 f k i k _t -t their
standard
0.191 0.0966 0.243 0.0935 0.326 0.153 0.340 0.247 0.351 0.275 0.638 0.450 0.734 0.555 0.600 0.560 0.980 0.587 1.05 0.699 1.78 1.05 errors
k 0.017 t 0.0201 k 0.016 -c 0.0294 -c 0.034 k 0.018 i 0.059 t 0.034 i 0.048 i 0.017 ” 0.094 AZ 0.010 k 0.143 + 0.077 k 0.101 k 0.087 * 0.164 ” 0.041 i 0.06 _f 0.044 k 0.37 2 0.08
x 10’ cpmig
2 SE)
(Mean
IN VARIOUS EXPERIMENTS
Skeletons
1
Lang
OF THE TRACER CONCENTRATIONS DURING THE SEQUENTIAL INJECTION
1.16 2.71 2.22 5.462 4.34 7.23 6.03 8.89 8.01 13.1 12.5 21.9 16.0 13.1 16.2 18.3 20.1 19.5 24.4 25.1 32.8 35.7
LEVELS
f 0.46 + 0.59 -CO.57 2 0.65 + 0.4 k 1.0 t 1.6 * 1.4 2 0.9 k 1.4 + 1.1 c 1.3 2 0.4 t- 1.4 k 4.0 k 2.9 z 1.6 2 0.9 + 3.7 _C 1.5 + 4.4 f 1.5
Liver
MEAN
and
i k -c r -+ k k + it t r k k -c + k t k ?L i _c lower
0.917 2.11 2.03 4.37 3.36 10.2 4.42 11.1 4.45 11.8 7.79 17.3 9.71 12.6 8.96 23.2 8.52 26.1 12.1 23.7 15.3 22.1
Gonads
values
0.270 0.22 0.23 0.40 0.55 2.7 0.47 0.9 0.31 1.5 0.76 1.4 0.86 1.6 1.31 4.1 0.66 3.9 1.4 3.8 1.6 4.7
ORGANS OF THE OF 109CdU
f 0.235 -
show
2.57
2.29
2.05
2.12
1.28
-
-
-
-
1.22 1.55 1.65 2.99 3.44 4.78 10.4 4.48 4.02 5.41 14.1 4.08 21.7 7.52 11.2 21.2 36.8 23.8
k
0.370
of live
females
2 0.44 2 0.34 k 0.27 k 1.05 -c 0.52 -+ 1.38 2 2.6 2 1.74 -c 0.87 2 0.88 ‘_ 3.6 k 1.22 -c 7.2 k 4.26 k 3.7 i 10.5 AC 10.2 e 2.3
-
-
Thymus
0.878
the average
k 0.38
c 0.35
k 0.43
t_ 0.98
t 0.19
0.815r0.198 0.828t0.156 1.03 ?z 0.20 0.870a0.068
0.885
0.562~0.164 -
glands
Prostate
MOUSE
With
0.348 0.305 0.507 0.316 0.665 0.555 0.702 0.560 0.981 0.804 1.35 1.27 1.77 1.44 1.57 1.62 1.79 1.89 2.04 2.57 4.07 3.13
their
-e 0.025 i 0.038 k 0.038 k 0.034 + 0.030 t 0.047 -c 0.095 k 0.047 -t 0.055 IT 0.067 -c0.080 -c 0.090 k 0.35 2 0.22 + 0.09 k 0.14 f 0.17 * 0.20 _c 0.40 k 0.26 + 0.67 AZ 0.54
Salivary glands
Cd
UPTAKE
IN
MOUSE
ORGANS
35
organs (Matsubara-Khan et al., to be published). This was deemed to be due to the limitation of analytical sensitivity. The fitness of Eq. (3) of the one compartment model for the data observed the uptake of various radionuclides from environmental water by marine organisms was confirmed broadly by Hiyama and Shimizu (1964) utilizing Cd, Cs, Sr, Zn and Ce and by Hiyama and Matsubara-Khan (1964) with 1, Fe, Co and Ru. They tried to compare the rate constants obtained from the single injection experiments with other rate constants obtained from sequential uptake experiments using the same organisms and found that in most radionuclides they were in good accord (Matsubara-Khan, I97 1). Hiyama and Shimizu (1964) had observed unique patterns in uptake of Cd and Zn from sea water by marine organisms which showed the curves with downward projection. They expected these patterns would fit the two-compartment model as Eq. (6). Thus we had expected turnover of ‘OgCd in mouse organs also should follow the kinetics of the two compartment-model and could observe a typical pattern of the two-compartment model in our previous data of single injection experiments obtained from kidney, liver and salivary glands as described before. The presently observed pattern of cadmium uptake in kidneys was sigmoidal, which seemed to be different from the ones in gonads. It was expected that the curvature might not be big due to slow speed of Cd turnover in kidney; however, in the observed patterns of other organs too the curvature was even smaller, nearly linear. The reason of a smaller rate of turnover in case of sequential uptake
j-&Cd I MOUSE WHOLE
BODY
80
,5'
,P'
,' 9'
o..O--L1 -\-- _
50
100 150 Go DAYS FIG. 3. Nordberg’s data of whole-body retention of cadmium in mouse after sequential oral intake of cadmium and provisionally fitted equation by the present authors. Data and figures were slightly modified by the authors. L 1: Exposed
to 0.5 mg “‘“Cd/kg
L2: Exposed
to 0.15 mg “‘“Cd/kg
body
body
weight
weight
5 days/week.
5 days/week,
36
MATSUBARA-KHAN
AND MACHIDA
; ..; .:o,,’ 4’ BONNELLet ;2,‘,,
al(1960)
30 60 90 120 150 MYS FIG. 4. Bonnell’s data of cadmium retention in the rat liver and kidneys during the sequential injections of cadmium, figures were slightly modified by the authors. 0.75 mg Cd/kg body weight were injected thrice a week.
KID.:
y ==m8.8
!I+
0.04 0.03 -0.03
e-n.o4*_
0.04 0.04 -0.03
e-“-03L! .
was not yet elucidated. The results showed us the necessity of longer experimental duration in order to observe the equilibrium of cadmium turnover at tracer level. Accordingly year-long experiments of sequential oral administration of cadmium to mice were carried out later on. Figure SA and B shows the results of time course study of cadmium accumulation in kidney or liver of mice fed a cadmium containing diet. The equilibrium was attained there 1 lo- 170 days after the start of continuous oral intake of cadmium, where a stable cadmium-added diet having the concentration of 2 ppm-wet was given to mice instead of the tracer injections. Moore et ul. (1973) have reported that the route of administration did not influence cadmium turnover at a late stage of the experiments. If we postulate that the equilibrium is attained about I50 days after the initiation of the present experiment in most organs, the organ levels at equilibrium are to be extrapolated. It was tried to calculate the theoretical curve of Eq. (6) using thus obtained equilibrium levels (u’/k) and rate constants obtained in the previous 1974a). Thus calculated theoretical research (k and k’ in Matsubara-Khan, curves are shown with broken lines on Fig. 2(A-D). The direct data fitting to Eq. (6) was impossible due to the freezing of data, since the curve was too linear due to their small rate constants. Also the duration of the experiments was too short to show the equilibrium point, though the parameter estimation by Deming’s (1964) least squares adjustments to Eq. (6) with computer system was working properly. For comparison’s sake it was provisionally tried to fit Eq. (6) with the data of cadmium retention by sequential uptake to the whole body of the mouse observed by Nordberg (1972) and with those in kidney and liver of rats reported by Bonnell et ai. (1960) (see Figs. 3 and 4). It is of interest that the estimated two rate constants in the whole body of the mouse in both levels of observations at higher (0.5) and lower (0.25) administrations were roughly of the same values. The fact that the accumulation pattern of cadmium even in the whole body
Cd UPTAKE Cd PpmMt
ACCUMULATION OBSERVED
37
IN MOUSE ORGANS IN MOUSE KIDNEY Bf AlOMlCAB%+TlON
SFECTRosIJ)F4
I 3. 1
15
50
110
Cd
v:
166
ACUIMULATION OBSERVED
269
IN M)USE BY AKIMK:
388
LIVER ABSORPTION
DAYS
SFECTFQXOPY
, 15
50
110
166
269
388 DAYS
FIG. 5. Stable cadmium accumulation in kidney (A) and liver (B) of the mouse as a result of oral intake of cadmium. Two parts per million-wet of cadmium containing mouse feed was given to 9 week-old-mice at the initiation of the experiment until the day of their autopsies. On the days given in the figures, five males and five females were autopsied and I3 organs were taken out. After ashing each organ by Trapelo LTA-30 1, cadmium was measured with a JarrehAsh AA780 atomic absorption spectroscope. (Detailed explanations will be given in a forthcoming paper.)
followed the two-compartment model means that turnover of cadmium in the organism does not fit the dynamics of simple one-compartment pattern in which the materials are taken up instantly and behave homogeneously. This seemed to be a reflexion of heterogeneous chemical states of cadmium in the organism. Since the fitness of the theoretical equations for the sequential uptake system to cadmium data was not as good as that of the single injection system, there must be more complicated mechanisms not accessible by simple models to cause skewing or variation, which is more prominent in the process of cadmium absorption by organisms. Also we cannot exclude the possibility of other explanations if the presence of a specific mechanism is physiologically verified, e.g., an increased excretion of cadmium due to tissue damage at later stage (Nordberg,
38
MATSUBARA-KHAN
AND MACHIDA
1972). Finally, we must emphasize that mathematical treatment was carried out not to prove the facts but to rearrange the obtained quantitative data so that we are able to try further quantitative estimation or to arrive at a new idea for a working hypothesis. REFERENCES Bonnell, J. A., Ross, J. H., and King, E. R. (1960). Renal lesions in experimental cadmium poisoning. Brit. J. Industr. Med. 17, 69-80. Cotzias, G. C., Borg, D. C., and Selleck, B. (1961). Virtual absence of turnover in cadmium metabolism: Cd’OS studies in the mouse. Amer. J. Physiol. 201, 927-930. Decker, L. E., Byerrum, R. U., Decker, C. F., Hoppert. C. A., and Langham. R. F. (1958). Chronic toxicity studies. I. Cadmium administered in drinking water to rats. AMA Arch. Industr. Health 18, 228-23 1. Deming, W. E. ( 1964). “Statistical Adjustment of Data.” Dover, New York. Hiyama, Y., and Shimizu, M. (1964). On the concentration factors of radioactive Cs. Sr, Cd, Zn and Ce in marine organisms. Rec. Oceanogruph. Works Jap. 7, 43-78. Hiyama, Y. and Matsubara-Khan, J. (1964). On the concentration factors of radioactive I, Co, Fe and Ru in marine organisms. Rec. Oceanograph. Works Jap. 7, 79-106. Matsubara-Khan, J. (197 I). Application of mathematical models in medical research-Examples and method of data adjustment. Igaku No Aywni 77, 625-635, 737-742 (in Japanese). Matsubara-Khan. J. (1974a). Compartmental analysis for the evaluation of biological half-lives of cadmium and mercury in mouse organs. Environ. Res. 7, 54-67. Matsubara-Khan, J., and Sei, K. (3974b). Evaluation of biological half-lives of radionuclides in mouse organs. Papers presented on the First World Congress of Nuclear Medicine. Tokyo, September 1974. Proceedings of the World Federation of Nuclear Medicine and Biology. Moore, W. J., Stara, J. F.. Cracker. W. C., Malanchuk, M.. and Iltis, R. (1973). Comparison of “jmcadmium retention in rats following different routes of administration. EmGron. Rrs. 6, 473-478. Nordberg, G. F. (1972). Cadmium metabolism and toxicity. Environ. Physiol. Biochem. 2, 7-36. Schroeder, H. A., and Balassa, J. J. (196 1). Abnormal trace metals in man: Cadmium. J. Chron. Dis. 14, 236-258.
RESEARCH
Cadmium
29-38
(1975)
Accumulation in Mouse Organs During Sequential Injections of Cadmium-109’
JUNKO School
10,
MATSUBARA-KHAN'AND qf Health
Sciences.
Faculty
KAZUHIKO of’ Medicine
the
MACHIDA
Uni\,rrsity
qf Tokyo.
Tokyo. Japnr1 Received
August
27. 1974
to demonstrate the accumulation Sequential injections of ‘OYCd on mice were performed patterns of cadmium in various organs in mammals at tracer level. During the experimental period of 130 days, slow and upward accumulation of the tracer continued in most organs except gonads in which the equilibrium was attained. These linear increments showed slightly downward projection as sigmoid, which suggested the fitness to the two-comamong difpartment-model. More than a thousandfold difference of ‘O”Cd concentrations ferent organs was observed, and these striking differences of tissue levels manifested the strong tissue specificity of cadmium distribution. Sexual variation was also demonstrated; in blood, stomach, liver, spleen and kidneys the tracer concentrations in females were from I. I to 1.5 times those of the males, while in the skeletons males kept a 1.5-fold higher concentration of the tracer.
INTRODUCTION
Virtual absence of cadmium turnover in the plasma of rat was reported by Cotzias (196 1). Schroeder ( 1961) noticed that cadmium behaves differently from other ubiquitous metals in mammals and is concentrated in the kidney against the zinc-cadmium concentration gradient. The unique character of cadmium turnover was further traced recently by Matsubara-Khan (1974a, 1974b) in the process of compartmental analysis of the data from the tracer experiments aimed at obtaining biological half lives of inorganic cadmium, mercury, zinc, chromium and selenium in various organs of mice. The quantitative behaviour of cadmium in animal organs can be characterized by the slow turnover as the data gave unusually small rate-constants and/or gave extremely long biological half-lives in comparison to other metals; thus in some organs data fitted the two-compartment model well, which suggested the presence of relatively compact metabolic barrier among the organs. Various reports on cadmium retention in mammalian tissues were mostly concerned about single administration experiments while data on cadmium accumulation during a sequential administration are scant. Decker ( 1958) observed linear increase of cadmium concentration in rat kidney and liver after continuous oral administration for 6 and 12 months. Accumulation patterns of cadmium in rat liver and kidney were also reported by Bonnell et al. (1960) and that of mouse whole body in the review of Nordberg (1972).
r All communications
regarding
this paper
may be sent to J. Matsubara-Khan 29
Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
30
MATSUBARA-KHAN
AND
MACHIDA
The present studies aimed at demonstrating the patterns of cadmium accumulation and the difference of concentration levels in various organs of mice as a result of prolonged sequential injections of radioactive cadmium in order to confirm their fitness to the compartment models for the sequential uptake experiments. MATHEMATICAL
MODELS
In the previous paper Matsubara-Khan (1974a, 1974b) the fitness of compartment models for the single injection systems at individual and organ levels of observations has been discussed, while this time discussions are concentrated on the application of compartment models for the sequential administration systems. The theoretical models are shown in Fig. 1 and underlying hypothesis and experimental situations were similar to the previous paper by the author, except that the tracer was sequentially injected twice a week up to the time of the autopsies. One-Compartment
Model for the Sequential
Irgection
Systems
The dynamics of the amounts of the tracer in an organ, which is regarded as one compartment q(t), is to be regulated by the rate of tracer uptake to the organ II, and the rate of tracer elimination which is the product of q(t) and a rate constant k. This relation is expressed as follows: -&W = M - k . q(t). dt
(1)
When u is constant, the integration of Eq. (I) between t = 0 and t = t with initial condition q(0) = 0 gives a solution, q(t) = f ( 1 - e-/i’t). If data are obtained as the relative substituted as follows:
concentration
(2) of the tracer, Eq. (2) can be
y(t) = z (1 - eCh”f)
B
t
up ’
9s (*I .[YW]
(3)
1
k’
I q(t)
/ .
[y(t)]
k I
4
FIG. I. The theoretical models for the turnover of the metal in the studies of sequential injections systems. For explanation see text. (A) One-compartment model in the sequential administration system. (B) Two-compartment model in the sequential administration system.
Cd
UPTAKE
IN
MOUSE
ORGANS
31
where y(t) is the concentration of tracer in organ, e.g., cpmlg wet tissue, provided that the observations are carried out using the animals in a steady state without noticeable growth. T\iso-Compar.tment Model.for the If the tracer reaches the organ ment, in a manner similar to the postulate the following equations second compartments,
Sequential Injection Systems under observation via a preceding compartone explained in the previous paper, we can for the amounts of tracer of the first and the
-dq’(t) = 11’ - A’ . q’(f) dt
-ddf) = k’ * q’(t) - k . q(t) dt where q’(t), ~1’ and I;’ are the amount of tracer, rate of uptake and rate constant of the first compartment and q(t) and li are those for the second ones, respectively. The solution of the above equations is given as follows with an initial condition that q’(O) = 0, q(0) = 0 at t = 0, 1 -k
k-k’
e -k-‘l
In terms of y(t) instead of q(t), we can get an equation similar to Eq. (6) where 11’ should be multiplied by the weight factor of both compartments, i.e.. W/W’. EXPERIMENTAL
Sixty male and 60 female ICR mice 8 weeks-of-age were utilized in the present study. With intervals of 3.5 days namely twice a week, carrier free ‘OsCd Cl, 6.5 &i/kg body weight was sequentially injected intraperitoneally to all animals until they were autopsied. At each time of injections the tracer was injected to each mouse with strict proportion to his body weight. Mice were kept at room temperature around 15°C and fed with ordinary mouse feed. Autopsies were carried out 11 times during the 130 days of experimental period. Each time five males and five females were sacrificed. Immediately afterwards, ten organs from each individual were taken, weighed and radioassayed with Aloka DC-6 well-type scintillation counter. RESULTS
Patterns of Cadmium
Accumulation
in Mouse Orguru
The concentrations of losCd, cpm/g wet tissue in each organ were plotted against time in days as shown in Fig. 2 (A-H). In each figure the open circles show the averages of data obtained from five females and closed circles show the average values from five males. From the results of single injection experiments described in the previous study ( 1974a), it was expected that the turnover speed of cadmium in kidneys and
Xl,O'CPM/y XlO'CPM/y ,%. A. KIDNEY 150
xlg+cPM /g 8. LlVER
loo100 . _.I
5050 & IIII, x1o*cPM/g g 1625 3544
I,,
5665
77
, , II,,, I I I I I 91 103DAysM) XlO+CPM/g 3 1625 35 44 56 $5 77 9'1 103mYS130
%
D. at%%
x20
III,, 916 25 3544
I,, 5665
77
, , I 91 103mys 130
1 I 9 169
I'll I1 b r 35 44 56 65 77 91 103D,&30
0
E. SPLEEN
91625 XlO+cPM&
3544
5665
77 $1 103 DAYS13 916 253544 0 X104CPMkJ
G. BONE 1.Q
0
.'o
0
l
5665
i7
91 103D4ys130 0
H. LUNG
8
0
6
0
/
45
916 25 35 44 56 65 77
91 103DAYS130
9 16 25 35 44 56 65 77
91 103DAys130
FIG. 2(A-H). Patterns of cadmium retention in various organs during the sequential injections of “Td to the mouse. Open circles show the averages of data obtained from five females, and closed circles show the average values from five males. Solid line shows the trend obtained from the averages of the observed data, while dotted line in each graph of A-D shows the theoretical curve of Eq. (6) calculated from the previously obtained rate constants in the single injection experiments and presently extrapolated equilibrium value. For detailed explanation see Discussion. Calculated equations
are as follows.
y = $
B 1 - __ B-C
e-et. ~ ‘B-C
C
where in graph A, kidney, were param>’ eters A = 1.24, B = 0.001. C = 0.30; in graph B, liver, A = 0.60, B = 0.01, C = 0.57; in graph C, salivary glands, A = 0.026. B = 0.002, C = 0.70: and in graph D, female gonad, A = 0.50. B=O.OlS, C= 0.17 and malegonad,A = 0.26, B =0.019, C=O.O52. (
32
e?r
Cd
UPTAKE
IN
MOUSE
ORGANS
33
salivary glands must be very slow because they gave extremely small rate constants. The present results showed that the turnover speed is slower not only in the above mentioned organs but also in most of the organs with the exception of gonads. In more than 4-months observation, slow upward accumulation of radiocadmium continued, only the data from gonads seemed to have attained steady state. These linear increments showed slightly downward projection. This pattern is quite different from the uptake pattern of other metals which fit the Eq. (3) of the one-compartment model having upward projection, hyperbolic curve. The accumulation pattern of ‘O”Cd in kidneys was sigmoidal (see Fig. 2A). This was quite in accord with our expectation, since our previous results of single injection data from kidneys had fitted the two compartment model. Tissue Concentration
Levels of “‘Cd
In Table 1 are shown the mean concentrations of ‘O”Cd from five males (upper) and from five females (below) in ten organs during the experimental period. Remarkable differences were observed in cadmium concentrations of various organs. On 130th day, the end of the experimental period, the relative concentration in kidneys was about 150; liver, 50; spleen, 30; uterus and ovary, 25; testis and seminal vesicles, 10; stomach or lung, 10; salivary glands or prostate glands, 3; skeletons, 1.5; pelt, 1.3: muscle, 0.6; brain, 0.1; and blood of males 0. 122 and blood of females l.6,2 respectively. These clear differences showed strong tissue specificity of cadmium distribution. Sexual Variation As the tracer was injected strictly proportional to the body weights of experimental animals, the data presented in Table 1 provide the comparison of the concentrations in males and females. It was noticed that females had generally higher concentrations. In blood, stomach, liver, spleen and kidney, the tracer concentrations in females was roughly 1. l- 1.5 times those of the males, while in the skeletons it was vice versa. DISCUSSION
More than a thousandfold difference of tracer concentrations among different organs of the mouse was observed. Highest concentration was observed in kidney with relative concentration of 150, while lowest one was in brain at 0.1. In these experiments, data had usually 20-40s of coefficients of variance. Standard errors of mean values were shown in Table 1, the data however manifested consistently clear differences of the tissue levels with repeated observations of eleven series of autopsies. This should be noticed as unique character of cadmium distribution which is different from zinc. Stable cadmium concentrations surveyed by atomic absorption spectroscopy in a separate study showed only hundredfold difference among the various ‘Nofr: Cadmium-109 in blood was assayedonly on the 130th day’s observation. Male average of five animals was 1924 cpm/g with standard error 572 and female averageof six animals was 23875 cPm/g with standard error 437 I. Though this difference was noteworthy, we could not confirm such noticeable sexual difference in a later study of atomic absorption spectroscopy; a confirmatory tracer experiment for this is not yet finished.
16
25
35
44
56
65
77
91
103
130
2
3
4
5
6
7
8
9
10
11
M F M F M F M F M F M F M F M F M F M F M F
Sex
’ 1a each column, standard errors.
9
Day
1
No.
Autopsy
c c * + t_ * 2 ? + i * i + + F + C f t &
upper
6.12-c 8.59~ 12.0 12.1 21.1 19.9 21.5 23.8 38.6 49.6 46.8 63.0 51.6 52.7 71.1 80.7 113.0 112.0 132.0 138.0 155.0 164.0
Kidney
values
0.64 1.15 1.2 1.2 1.7 1.2 1.9 1.8 2.2 11.9 4.1 3.8 6.5 5.4 9.9 7.1 8.0 9.0 12.0 15.0 15.0 14.0
show
4.74 6.63 7.61 8.96 9.45 11.3 12.4 14.5 14.3 15.3 15.8 21.1 12.7 14.8 20.8 32.1 22.5 36.1 32.4 38.1 36.0 51.0
the average
TABLE
tracer
t 0.25 i 0.32 k 0.14 1.13 k 0.71 k 0.63 i 1.22 -+ 0.88 k 1.17 k2.1 k 1.7 ~3.6 k 1.7 kO.8 i- 2.3 k 3.2 ~3.8 24.8 k5.8 -c3.3 k6.2 i3.3
Spleen
k t t_ k k 2 k k t -t k ?I -c f t k k t -t _f i 2
concentrations
0.491 1.29 0.810 2.13 1.46 2.04 1.95 3.38 2.26 5.53 2.61 5.02 4.55 6.85 3.43 6.83 3.35 6.45 6.93 9.82 7.40 12.3
Stomach
0.235 0.298 1.04 0.765 0.784 0.594 1.61 0.748 1.93 1.13 2.72 2.50 4.83 1.86 4.40 4.35 2.72 3.30 4.30 6.99 5.43 8.41
of 5 males
0.107 0.16 0.068 0.20 0.25 0.11 0.30 0.54 0.35 0.78 0.25 0.82 0.37 0.59 0.61 1.78 0.55 1.11 0.70 2.56 0.23 11.1
0.017 0.051 0.40 0.329 0.163 0.124 0.39 0.075 0.72 0.12 0.94 0.88 1.52 0.12 1.19 1.57 0.27 0.89 0.52 3.07 1.70 1.97 with
k + _f f_ k t 2 t + + t ik t -c 2 f k i k _t -t their
standard
0.191 0.0966 0.243 0.0935 0.326 0.153 0.340 0.247 0.351 0.275 0.638 0.450 0.734 0.555 0.600 0.560 0.980 0.587 1.05 0.699 1.78 1.05 errors
k 0.017 t 0.0201 k 0.016 -c 0.0294 -c 0.034 k 0.018 i 0.059 t 0.034 i 0.048 i 0.017 ” 0.094 AZ 0.010 k 0.143 + 0.077 k 0.101 k 0.087 * 0.164 ” 0.041 i 0.06 _f 0.044 k 0.37 2 0.08
x 10’ cpmig
2 SE)
(Mean
IN VARIOUS EXPERIMENTS
Skeletons
1
Lang
OF THE TRACER CONCENTRATIONS DURING THE SEQUENTIAL INJECTION
1.16 2.71 2.22 5.462 4.34 7.23 6.03 8.89 8.01 13.1 12.5 21.9 16.0 13.1 16.2 18.3 20.1 19.5 24.4 25.1 32.8 35.7
LEVELS
f 0.46 + 0.59 -CO.57 2 0.65 + 0.4 k 1.0 t 1.6 * 1.4 2 0.9 k 1.4 + 1.1 c 1.3 2 0.4 t- 1.4 k 4.0 k 2.9 z 1.6 2 0.9 + 3.7 _C 1.5 + 4.4 f 1.5
Liver
MEAN
and
i k -c r -+ k k + it t r k k -c + k t k ?L i _c lower
0.917 2.11 2.03 4.37 3.36 10.2 4.42 11.1 4.45 11.8 7.79 17.3 9.71 12.6 8.96 23.2 8.52 26.1 12.1 23.7 15.3 22.1
Gonads
values
0.270 0.22 0.23 0.40 0.55 2.7 0.47 0.9 0.31 1.5 0.76 1.4 0.86 1.6 1.31 4.1 0.66 3.9 1.4 3.8 1.6 4.7
ORGANS OF THE OF 109CdU
f 0.235 -
show
2.57
2.29
2.05
2.12
1.28
-
-
-
-
1.22 1.55 1.65 2.99 3.44 4.78 10.4 4.48 4.02 5.41 14.1 4.08 21.7 7.52 11.2 21.2 36.8 23.8
k
0.370
of live
females
2 0.44 2 0.34 k 0.27 k 1.05 -c 0.52 -+ 1.38 2 2.6 2 1.74 -c 0.87 2 0.88 ‘_ 3.6 k 1.22 -c 7.2 k 4.26 k 3.7 i 10.5 AC 10.2 e 2.3
-
-
Thymus
0.878
the average
k 0.38
c 0.35
k 0.43
t_ 0.98
t 0.19
0.815r0.198 0.828t0.156 1.03 ?z 0.20 0.870a0.068
0.885
0.562~0.164 -
glands
Prostate
MOUSE
With
0.348 0.305 0.507 0.316 0.665 0.555 0.702 0.560 0.981 0.804 1.35 1.27 1.77 1.44 1.57 1.62 1.79 1.89 2.04 2.57 4.07 3.13
their
-e 0.025 i 0.038 k 0.038 k 0.034 + 0.030 t 0.047 -c 0.095 k 0.047 -t 0.055 IT 0.067 -c0.080 -c 0.090 k 0.35 2 0.22 + 0.09 k 0.14 f 0.17 * 0.20 _c 0.40 k 0.26 + 0.67 AZ 0.54
Salivary glands
Cd
UPTAKE
IN
MOUSE
ORGANS
35
organs (Matsubara-Khan et al., to be published). This was deemed to be due to the limitation of analytical sensitivity. The fitness of Eq. (3) of the one compartment model for the data observed the uptake of various radionuclides from environmental water by marine organisms was confirmed broadly by Hiyama and Shimizu (1964) utilizing Cd, Cs, Sr, Zn and Ce and by Hiyama and Matsubara-Khan (1964) with 1, Fe, Co and Ru. They tried to compare the rate constants obtained from the single injection experiments with other rate constants obtained from sequential uptake experiments using the same organisms and found that in most radionuclides they were in good accord (Matsubara-Khan, I97 1). Hiyama and Shimizu (1964) had observed unique patterns in uptake of Cd and Zn from sea water by marine organisms which showed the curves with downward projection. They expected these patterns would fit the two-compartment model as Eq. (6). Thus we had expected turnover of ‘OgCd in mouse organs also should follow the kinetics of the two compartment-model and could observe a typical pattern of the two-compartment model in our previous data of single injection experiments obtained from kidney, liver and salivary glands as described before. The presently observed pattern of cadmium uptake in kidneys was sigmoidal, which seemed to be different from the ones in gonads. It was expected that the curvature might not be big due to slow speed of Cd turnover in kidney; however, in the observed patterns of other organs too the curvature was even smaller, nearly linear. The reason of a smaller rate of turnover in case of sequential uptake
j-&Cd I MOUSE WHOLE
BODY
80
,5'
,P'
,' 9'
o..O--L1 -\-- _
50
100 150 Go DAYS FIG. 3. Nordberg’s data of whole-body retention of cadmium in mouse after sequential oral intake of cadmium and provisionally fitted equation by the present authors. Data and figures were slightly modified by the authors. L 1: Exposed
to 0.5 mg “‘“Cd/kg
L2: Exposed
to 0.15 mg “‘“Cd/kg
body
body
weight
weight
5 days/week.
5 days/week,
36
MATSUBARA-KHAN
AND MACHIDA
; ..; .:o,,’ 4’ BONNELLet ;2,‘,,
al(1960)
30 60 90 120 150 MYS FIG. 4. Bonnell’s data of cadmium retention in the rat liver and kidneys during the sequential injections of cadmium, figures were slightly modified by the authors. 0.75 mg Cd/kg body weight were injected thrice a week.
KID.:
y ==m8.8
!I+
0.04 0.03 -0.03
e-n.o4*_
0.04 0.04 -0.03
e-“-03L! .
was not yet elucidated. The results showed us the necessity of longer experimental duration in order to observe the equilibrium of cadmium turnover at tracer level. Accordingly year-long experiments of sequential oral administration of cadmium to mice were carried out later on. Figure SA and B shows the results of time course study of cadmium accumulation in kidney or liver of mice fed a cadmium containing diet. The equilibrium was attained there 1 lo- 170 days after the start of continuous oral intake of cadmium, where a stable cadmium-added diet having the concentration of 2 ppm-wet was given to mice instead of the tracer injections. Moore et ul. (1973) have reported that the route of administration did not influence cadmium turnover at a late stage of the experiments. If we postulate that the equilibrium is attained about I50 days after the initiation of the present experiment in most organs, the organ levels at equilibrium are to be extrapolated. It was tried to calculate the theoretical curve of Eq. (6) using thus obtained equilibrium levels (u’/k) and rate constants obtained in the previous 1974a). Thus calculated theoretical research (k and k’ in Matsubara-Khan, curves are shown with broken lines on Fig. 2(A-D). The direct data fitting to Eq. (6) was impossible due to the freezing of data, since the curve was too linear due to their small rate constants. Also the duration of the experiments was too short to show the equilibrium point, though the parameter estimation by Deming’s (1964) least squares adjustments to Eq. (6) with computer system was working properly. For comparison’s sake it was provisionally tried to fit Eq. (6) with the data of cadmium retention by sequential uptake to the whole body of the mouse observed by Nordberg (1972) and with those in kidney and liver of rats reported by Bonnell et ai. (1960) (see Figs. 3 and 4). It is of interest that the estimated two rate constants in the whole body of the mouse in both levels of observations at higher (0.5) and lower (0.25) administrations were roughly of the same values. The fact that the accumulation pattern of cadmium even in the whole body
Cd UPTAKE Cd PpmMt
ACCUMULATION OBSERVED
37
IN MOUSE ORGANS IN MOUSE KIDNEY Bf AlOMlCAB%+TlON
SFECTRosIJ)F4
I 3. 1
15
50
110
Cd
v:
166
ACUIMULATION OBSERVED
269
IN M)USE BY AKIMK:
388
LIVER ABSORPTION
DAYS
SFECTFQXOPY
, 15
50
110
166
269
388 DAYS
FIG. 5. Stable cadmium accumulation in kidney (A) and liver (B) of the mouse as a result of oral intake of cadmium. Two parts per million-wet of cadmium containing mouse feed was given to 9 week-old-mice at the initiation of the experiment until the day of their autopsies. On the days given in the figures, five males and five females were autopsied and I3 organs were taken out. After ashing each organ by Trapelo LTA-30 1, cadmium was measured with a JarrehAsh AA780 atomic absorption spectroscope. (Detailed explanations will be given in a forthcoming paper.)
followed the two-compartment model means that turnover of cadmium in the organism does not fit the dynamics of simple one-compartment pattern in which the materials are taken up instantly and behave homogeneously. This seemed to be a reflexion of heterogeneous chemical states of cadmium in the organism. Since the fitness of the theoretical equations for the sequential uptake system to cadmium data was not as good as that of the single injection system, there must be more complicated mechanisms not accessible by simple models to cause skewing or variation, which is more prominent in the process of cadmium absorption by organisms. Also we cannot exclude the possibility of other explanations if the presence of a specific mechanism is physiologically verified, e.g., an increased excretion of cadmium due to tissue damage at later stage (Nordberg,
38
MATSUBARA-KHAN
AND MACHIDA
1972). Finally, we must emphasize that mathematical treatment was carried out not to prove the facts but to rearrange the obtained quantitative data so that we are able to try further quantitative estimation or to arrive at a new idea for a working hypothesis. REFERENCES Bonnell, J. A., Ross, J. H., and King, E. R. (1960). Renal lesions in experimental cadmium poisoning. Brit. J. Industr. Med. 17, 69-80. Cotzias, G. C., Borg, D. C., and Selleck, B. (1961). Virtual absence of turnover in cadmium metabolism: Cd’OS studies in the mouse. Amer. J. Physiol. 201, 927-930. Decker, L. E., Byerrum, R. U., Decker, C. F., Hoppert. C. A., and Langham. R. F. (1958). Chronic toxicity studies. I. Cadmium administered in drinking water to rats. AMA Arch. Industr. Health 18, 228-23 1. Deming, W. E. ( 1964). “Statistical Adjustment of Data.” Dover, New York. Hiyama, Y., and Shimizu, M. (1964). On the concentration factors of radioactive Cs. Sr, Cd, Zn and Ce in marine organisms. Rec. Oceanogruph. Works Jap. 7, 43-78. Hiyama, Y. and Matsubara-Khan, J. (1964). On the concentration factors of radioactive I, Co, Fe and Ru in marine organisms. Rec. Oceanograph. Works Jap. 7, 79-106. Matsubara-Khan, J. (197 I). Application of mathematical models in medical research-Examples and method of data adjustment. Igaku No Aywni 77, 625-635, 737-742 (in Japanese). Matsubara-Khan. J. (1974a). Compartmental analysis for the evaluation of biological half-lives of cadmium and mercury in mouse organs. Environ. Res. 7, 54-67. Matsubara-Khan, J., and Sei, K. (3974b). Evaluation of biological half-lives of radionuclides in mouse organs. Papers presented on the First World Congress of Nuclear Medicine. Tokyo, September 1974. Proceedings of the World Federation of Nuclear Medicine and Biology. Moore, W. J., Stara, J. F.. Cracker. W. C., Malanchuk, M.. and Iltis, R. (1973). Comparison of “jmcadmium retention in rats following different routes of administration. EmGron. Rrs. 6, 473-478. Nordberg, G. F. (1972). Cadmium metabolism and toxicity. Environ. Physiol. Biochem. 2, 7-36. Schroeder, H. A., and Balassa, J. J. (196 1). Abnormal trace metals in man: Cadmium. J. Chron. Dis. 14, 236-258.
