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Long term lung clearance and cellular retention of cadmium in rats and monkeys
J. Aerosol Sci., Vol. 18. No. 6, pp. 745-748, 1987.
0021-8502/87 $3.00+0.00 Pergamon Journals Ltd.
Printed in Great Britain
LONG TERM LUNG CLEARANCE AND CELLULAR RETENTION OF CADMIUM IN RATS AND MONKEYS G. 0berdorster, C. Cox and R. Baggs University of Rochester School of Medicine and Dentistry Rochester, NY 14642
Introduction
Results of both experimental animal studies and of epidemiologieal studies have shown that chronically inhaled cadmium compounds can have deleterious effects. Lung emphysema, lung fibrosis, chronic bronchitis, lung tumors and also renal dysfunction have been described (Friberg et al, 1974; Holden, 1980; Takenaka et al, 1983; Thun et al, 1985). Most of the experimental animal studies have been performed in rats, and the cadmium compound used was mostly CdCI 9. A more relevant compound for occupational human exposure, however, is Cd0. Thus,'when attempting to extrapolate results from rat inhalation studies with CdCI 9 to humans the question of the validity of such attempts arises. For example, w e % a v e found that CdCI 9 inhaled at low concentrations induces primary lung tumors in rats (Takenaka et ~i, 1983) yet epidemiological evidence in Cd exposed human workers seems to indicate a lower sensitivity of humans towards the pulmonary carcinogenic effect of inhaled Cd. An important factor for pulmonary effects of an inhaled substance is its retention in the lung. We have previously found, that both inhaled CdCI~ and CdO have a pulmonary retention half-time of about 70 days in Wistar-rats (O~erdorster et al, 1979). Our present studies were aimed at determining pulmonary retention of inhaled CdCI 9 in monkeys - reasoning that results are more easily transferable to humans and can %e used for extrapolation modelling - and in Long Evans rats, a different rat strain than used in our previous experiments. In addition, one monkey was also exposed to CdO.
Methods leo Thirty male Long Evans rats, b.w. about 220g, were exposed in nose only tubes to v-CdCl 2 aerosols generated by a jet nebulizer for 45 min. The activity median aerodynamic diameter (AMAD) of the particles was 0.23 m with a geometric standaq[d deviation (GSD) of 1.6, the cadmium concentration of the inhaled aerosol was 73 g/m ". Two female Macaca f a ~ c u l a r i s monkeys (b.w. 2.5 and 2.8 kg) were exposed via endotracheal tubes to ~v~CdCl2 aerosols, generated by an ultrasonic nebulizer, for 45 min. The monkeys were anesthetized and an artificial respiratory cycle was used with a 6 second inspiratory pause to increase lung deposition of the aerosol. AMAD was 2.5 um with a GSD of 3.6 for one monkey, and for the other monkey AMAD was 0.29 m with a GSD of 2.5 (use of baffle system to retain large pjrticles in ultrasonic generator). ~ective Cd conce~rqc~,~ions were 906 and 637 ug/m . A third monkey was e x p o ~ to .... Cd0 particles. ~ ....Cd0 had been produced by neutron irradiation of stable ~'~Cd0 particles. The particles were suspended in water and nebulized with an ultrasonic nebulizer, r e s ~ n g in an AMAD of 1.6 m and GSD of 2.5. Because of the low specific activity of .... CdO, exposure concentration had to be increased to 8.6 mg/m . However, no acute toxic effects on the respiratory system were detectable after the 45 min exposure or on the following days. In both rats and monkeys thoracic activity of the respective Cd isotopes was counted frequently up to day 240 (rats) and day 650 (CdCI 9 monkey) or day 240 (Cd0 monkey). A collimated double detector counting system was Used which focused on the lung only, excluding liver activity. In the rat ~ d y , animals were additionally killed on days 1,7,14,30,60,120 and 240 to measure v'Cd activity in organs. Renal 109Cd and llSmcd activity was also determined noninvasively in the monkeys by counting the region of the left (lower) kidney with a collimated system. After the end of the counting period, the monkeys were killed with an overdose of i.v.-administered pentobarbital and organ activities were measured. Lung sections were prepared for autoradiography, using Kodak dipping emulsion and exposing the applied emulsion for 6 months followed by counterstaining the sections with H.E.
745
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I
I
250
I
I:E
-6
o
10
20
100
50
150
Days a f t e r l n h o l o t i o n
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,
o
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,
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I 600
oI
Fig 3: Accumulation o [ 1 0 9 C d in k i d n e y s macaca fasci~H~aris monkeys after single exposure to - ~ - C d C I 2 on day O.
500
1.000
2,000
5.000
cpm '
,
0D
800
of 2 45 min
°
2:
2.5
y -
,
Pulmonary
•
o o
.
o
I 400
o°
I
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o
y=9341
,
retention
of
t
-
o
I 600
o
800
Fig 4: Autoradiographic tracheal caLtilage labelling 65~n~ays after single 45 min exposure to -v-CdCl 2 aerosols.
monkeys after
( C,d]l~
4 - - - -
109
o
_ln2~ C e 7~
inhaled
Days After Exposure
964
o ~
I 200
o
I
o ooO
5602 " e
.
aerosols in 2 macaca fasclcular~s single 45 min exposure on day O.
Fig
I
i
10 ~1~°~o 0
cpm xlO-3|
~-.
y = 343 • e
I
50
In 2 ,~. 85
I
~ -~ o
t
100-
200
I
Fig I: Pulmonary retention of inhaled I09CdC12 aerosols in Long Evans rats after single 45 mln exposure on day O.
I (,.)
0
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o
E
500
>
© X
~D
m
F~ ;= ©
©
--I C~
Long term lung clearance and cell retention of cadmium
747
Results Lung retention of inhaled I09cdC19 in the rats could be described by a monoexponential function from which a rete%tion halftime of 85 days could be determined (figure I, corrected for radioactive decay). Both nonlnvasive thoracic counting and counting of t~9excised lungs gave essentially the same results, In the monkeys, lung retention of CdCl 9 showed retention halftimes of 736 and 964 days, which were not significantly different (fig 2; nonlinear analysis of covariance). Accumulation in ~ _ kidney showed a steady increase over the time measured (fig 3). Lung retention of --'Cd0 s ~ e d a half time of 637 days, w5% ~ was also n%t significantly different from the CdCI 2 retention data. However, Cd in the Kianey snowea a steaay state level after about day 50. No signs of renal damage could be detected by clinical urinalysis or histology o f the kidney. The autoradiographic measurements showed significant labelling of the tracheal cartilage (Fig 4), probably connected with chondrocytes. In the interstitium of the lung, interstitial macrophages carried the highest amount of label, whereas alveolar epithelial cells showed less activity. Discussion We could show in these studies that rats and monkeys retain inhaled cadmium with significant different pulmonary retention half times. Since monkeys are closer related to humans, we suggest to apply the longer retention half time found in the monkeys when modeling lung Cd retention in humans. We confirmed our earlier findings that retention halftimes of Cd0 and CdC19are not significantly different from each other. This is probably due to the fact that Cd0 particles are rapidly solubilized in the lung (Hadley et al, 1980) possibly in the phagolysosomes of alveolar macrophages (Lundborg et al, 1985) and the ionic Cd is then retained in the lung bound to protein. This binding could include metallothionein which is induced in the lung by inhalation of Cd (Oberdorster and Kordel, 1981; Post et al, 1982), probably also in pulmonary macrophages (Iijima, Y. et al 1987). Possibly, differences in metallothionein metabolism and/or macrophage turnover times between rats and monkeys could explain the different pulmonary retention halftimes. Our autoradiographic results seem to indicate that interstitial macrophages more than alveolar epithelial cells are sites of pulmonary Cd storage. This indicates that initially cadmium deposited by inhalation in the alveolar space could be retained in alveolar macrophages and alveolar epithelial cells and is then slowly transferred to the interstitium, where it is retained mostly in interstitial macrophages. Further transport either via direct uptake into the blood compartment or indirectly via the lymphatic system into body organs including liver and kidney follows. Whether accumulation in the tracheal cartilage represents transfer of Cd from the low tracheobronchial circulation or whether it is due to tracheal uptake cannot be derived from our studies. We have no evidence favoring one or the other mechanism. The surprising finding of tracheal cartilage labelling - indicating Cd accumulation in this structure - is similar to a previously reported accumulation of inhaled Co-compounds in cartilage of trachea and bronchi in dogs (Kreyling, et al., 1986). The significance of such findings is not known at present. The continuing increase of Cd in the kidney even 2 years after a single 45 min inhalation exposure to Cd aerosols points out that I. lung tissue Cd acts as an important pool for C d accumulation in the kidney and 2. the primate kidney retains Cd with a very long retention halftime, as already described frequently (e.g. Friberg et al, 1974). It can easily be envisioned that chronic or repeated inhalation exposure to cadmium leads eventually to high renal Cd burdens. We conclude, that extrapolation to humans of results from chronic inhalation studies with Cd in rodents have to be made with caution since during chronic Cd inhalation exposure Cd will accumulate differently in the lungs of rats and primates. Acknowledgements The authors are grateful for the excellent technical assistance of Ms. N. Corson and S. Drago. This work is based partially on NIH grants ES01247 and ES01248. References Friberg, L., Piscator, M., Nordberg, G.F. and Kjellstrom, T. (1974) "Cadmium in the environment". Second edition, CRC Press, Cleveland, Ohio. Hadley, J.G., Conklin, A.M., Sanders, C.L. (1980) "Rapid solubilization and translocation of 109-Cd0 following pulmonary deposition". Toxicol. Appl. Pharmacol. 54:156-160.
748
G. OBERDORSTER, C. Cox and R. BAGGS
Holden, H. (1980) "Cadmium and pulmonary emphysema". Lancet 1:1137. lijima, Y., Takahashi, T., Fukushlma, T., Abe, S., llano, Y. and Kosaka, F. (1987) "Induction of metallothioneln by a macrophage factor and the partial characterization of the factor." Toxlcol. AppI. Pharmacol. 89:135-140. Kreyling, W.G., Bloom, S.D. and Brain, J.D. (1986) The uptake of soluble cobalt in the extrapulmonary airways of hamsters. In: Deposition and Clearance of Aerosols in the Human Respiratory Tract. Ed. W. Hoffmann, Facultas Universitatsverlag, Salzburg, pp. 62-77. Lundborg, M., Ling, B., Camner, P. (1984) "Ability of rabbit alveolar macrophages to dissolve metals". Exp. Lung Res. 7:11-22. Oberdorster, G., Baumert, H.P., Hochrainer, D., Stober, W. (1979) "The clearance of cadmium aerosols after inhalation exposure". Am. Ind. H~g. Assoc. J. 40:443-450. Oberdorster, kidney after Consultants. Environment.
G. and Kordel, W. (1981) "Metallothionein content in lung, liver, and chronic cadmium oxide and zinc oxide inhalation in rats." In: CEP Proceedings of the International Conference on Heavy Metals in the Edinburgh, pp. ~"0-~-5~5.
Post, C.T., Squibb, K.S., Fowler, B.A., Gardner, D.E., Illing, J., Hook, G.E.R. (1982) "Production of low molecular weight cadmium binding protein in rabbit lungs following exposure to cadmium chloride. Biochem. Pharmacol. 31:2969-2975. Takenaka, S., Oldiges, H., Konig, H., Hochrainer, H. and 0berdorster, G. (1983) "Carcinogenicity of cadmium chloride aerosols in Wistar rats. J. Natl. Cancer Inst. 70:367-373.
Thun, M.T., Schnorr, T.M., Smith, A.B., Halperin, W.E. and Lemen, R.A. (1985) "Mortality among a cohort of US cadmium production workers - An update". J. Natl. Cancer Inst. 74:325-333.
0021-8502/87 $3.00+0.00 Pergamon Journals Ltd.
Printed in Great Britain
LONG TERM LUNG CLEARANCE AND CELLULAR RETENTION OF CADMIUM IN RATS AND MONKEYS G. 0berdorster, C. Cox and R. Baggs University of Rochester School of Medicine and Dentistry Rochester, NY 14642
Introduction
Results of both experimental animal studies and of epidemiologieal studies have shown that chronically inhaled cadmium compounds can have deleterious effects. Lung emphysema, lung fibrosis, chronic bronchitis, lung tumors and also renal dysfunction have been described (Friberg et al, 1974; Holden, 1980; Takenaka et al, 1983; Thun et al, 1985). Most of the experimental animal studies have been performed in rats, and the cadmium compound used was mostly CdCI 9. A more relevant compound for occupational human exposure, however, is Cd0. Thus,'when attempting to extrapolate results from rat inhalation studies with CdCI 9 to humans the question of the validity of such attempts arises. For example, w e % a v e found that CdCI 9 inhaled at low concentrations induces primary lung tumors in rats (Takenaka et ~i, 1983) yet epidemiological evidence in Cd exposed human workers seems to indicate a lower sensitivity of humans towards the pulmonary carcinogenic effect of inhaled Cd. An important factor for pulmonary effects of an inhaled substance is its retention in the lung. We have previously found, that both inhaled CdCI~ and CdO have a pulmonary retention half-time of about 70 days in Wistar-rats (O~erdorster et al, 1979). Our present studies were aimed at determining pulmonary retention of inhaled CdCI 9 in monkeys - reasoning that results are more easily transferable to humans and can %e used for extrapolation modelling - and in Long Evans rats, a different rat strain than used in our previous experiments. In addition, one monkey was also exposed to CdO.
Methods leo Thirty male Long Evans rats, b.w. about 220g, were exposed in nose only tubes to v-CdCl 2 aerosols generated by a jet nebulizer for 45 min. The activity median aerodynamic diameter (AMAD) of the particles was 0.23 m with a geometric standaq[d deviation (GSD) of 1.6, the cadmium concentration of the inhaled aerosol was 73 g/m ". Two female Macaca f a ~ c u l a r i s monkeys (b.w. 2.5 and 2.8 kg) were exposed via endotracheal tubes to ~v~CdCl2 aerosols, generated by an ultrasonic nebulizer, for 45 min. The monkeys were anesthetized and an artificial respiratory cycle was used with a 6 second inspiratory pause to increase lung deposition of the aerosol. AMAD was 2.5 um with a GSD of 3.6 for one monkey, and for the other monkey AMAD was 0.29 m with a GSD of 2.5 (use of baffle system to retain large pjrticles in ultrasonic generator). ~ective Cd conce~rqc~,~ions were 906 and 637 ug/m . A third monkey was e x p o ~ to .... Cd0 particles. ~ ....Cd0 had been produced by neutron irradiation of stable ~'~Cd0 particles. The particles were suspended in water and nebulized with an ultrasonic nebulizer, r e s ~ n g in an AMAD of 1.6 m and GSD of 2.5. Because of the low specific activity of .... CdO, exposure concentration had to be increased to 8.6 mg/m . However, no acute toxic effects on the respiratory system were detectable after the 45 min exposure or on the following days. In both rats and monkeys thoracic activity of the respective Cd isotopes was counted frequently up to day 240 (rats) and day 650 (CdCI 9 monkey) or day 240 (Cd0 monkey). A collimated double detector counting system was Used which focused on the lung only, excluding liver activity. In the rat ~ d y , animals were additionally killed on days 1,7,14,30,60,120 and 240 to measure v'Cd activity in organs. Renal 109Cd and llSmcd activity was also determined noninvasively in the monkeys by counting the region of the left (lower) kidney with a collimated system. After the end of the counting period, the monkeys were killed with an overdose of i.v.-administered pentobarbital and organ activities were measured. Lung sections were prepared for autoradiography, using Kodak dipping emulsion and exposing the applied emulsion for 6 months followed by counterstaining the sections with H.E.
745
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10
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Days a f t e r l n h o l o t i o n
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,
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Fig 3: Accumulation o [ 1 0 9 C d in k i d n e y s macaca fasci~H~aris monkeys after single exposure to - ~ - C d C I 2 on day O.
500
1.000
2,000
5.000
cpm '
,
0D
800
of 2 45 min
°
2:
2.5
y -
,
Pulmonary
•
o o
.
o
I 400
o°
I
•-
o
y=9341
,
retention
of
t
-
o
I 600
o
800
Fig 4: Autoradiographic tracheal caLtilage labelling 65~n~ays after single 45 min exposure to -v-CdCl 2 aerosols.
monkeys after
( C,d]l~
4 - - - -
109
o
_ln2~ C e 7~
inhaled
Days After Exposure
964
o ~
I 200
o
I
o ooO
5602 " e
.
aerosols in 2 macaca fasclcular~s single 45 min exposure on day O.
Fig
I
i
10 ~1~°~o 0
cpm xlO-3|
~-.
y = 343 • e
I
50
In 2 ,~. 85
I
~ -~ o
t
100-
200
I
Fig I: Pulmonary retention of inhaled I09CdC12 aerosols in Long Evans rats after single 45 mln exposure on day O.
I (,.)
0
u
o
E
500
>
© X
~D
m
F~ ;= ©
©
--I C~
Long term lung clearance and cell retention of cadmium
747
Results Lung retention of inhaled I09cdC19 in the rats could be described by a monoexponential function from which a rete%tion halftime of 85 days could be determined (figure I, corrected for radioactive decay). Both nonlnvasive thoracic counting and counting of t~9excised lungs gave essentially the same results, In the monkeys, lung retention of CdCl 9 showed retention halftimes of 736 and 964 days, which were not significantly different (fig 2; nonlinear analysis of covariance). Accumulation in ~ _ kidney showed a steady increase over the time measured (fig 3). Lung retention of --'Cd0 s ~ e d a half time of 637 days, w5% ~ was also n%t significantly different from the CdCI 2 retention data. However, Cd in the Kianey snowea a steaay state level after about day 50. No signs of renal damage could be detected by clinical urinalysis or histology o f the kidney. The autoradiographic measurements showed significant labelling of the tracheal cartilage (Fig 4), probably connected with chondrocytes. In the interstitium of the lung, interstitial macrophages carried the highest amount of label, whereas alveolar epithelial cells showed less activity. Discussion We could show in these studies that rats and monkeys retain inhaled cadmium with significant different pulmonary retention half times. Since monkeys are closer related to humans, we suggest to apply the longer retention half time found in the monkeys when modeling lung Cd retention in humans. We confirmed our earlier findings that retention halftimes of Cd0 and CdC19are not significantly different from each other. This is probably due to the fact that Cd0 particles are rapidly solubilized in the lung (Hadley et al, 1980) possibly in the phagolysosomes of alveolar macrophages (Lundborg et al, 1985) and the ionic Cd is then retained in the lung bound to protein. This binding could include metallothionein which is induced in the lung by inhalation of Cd (Oberdorster and Kordel, 1981; Post et al, 1982), probably also in pulmonary macrophages (Iijima, Y. et al 1987). Possibly, differences in metallothionein metabolism and/or macrophage turnover times between rats and monkeys could explain the different pulmonary retention halftimes. Our autoradiographic results seem to indicate that interstitial macrophages more than alveolar epithelial cells are sites of pulmonary Cd storage. This indicates that initially cadmium deposited by inhalation in the alveolar space could be retained in alveolar macrophages and alveolar epithelial cells and is then slowly transferred to the interstitium, where it is retained mostly in interstitial macrophages. Further transport either via direct uptake into the blood compartment or indirectly via the lymphatic system into body organs including liver and kidney follows. Whether accumulation in the tracheal cartilage represents transfer of Cd from the low tracheobronchial circulation or whether it is due to tracheal uptake cannot be derived from our studies. We have no evidence favoring one or the other mechanism. The surprising finding of tracheal cartilage labelling - indicating Cd accumulation in this structure - is similar to a previously reported accumulation of inhaled Co-compounds in cartilage of trachea and bronchi in dogs (Kreyling, et al., 1986). The significance of such findings is not known at present. The continuing increase of Cd in the kidney even 2 years after a single 45 min inhalation exposure to Cd aerosols points out that I. lung tissue Cd acts as an important pool for C d accumulation in the kidney and 2. the primate kidney retains Cd with a very long retention halftime, as already described frequently (e.g. Friberg et al, 1974). It can easily be envisioned that chronic or repeated inhalation exposure to cadmium leads eventually to high renal Cd burdens. We conclude, that extrapolation to humans of results from chronic inhalation studies with Cd in rodents have to be made with caution since during chronic Cd inhalation exposure Cd will accumulate differently in the lungs of rats and primates. Acknowledgements The authors are grateful for the excellent technical assistance of Ms. N. Corson and S. Drago. This work is based partially on NIH grants ES01247 and ES01248. References Friberg, L., Piscator, M., Nordberg, G.F. and Kjellstrom, T. (1974) "Cadmium in the environment". Second edition, CRC Press, Cleveland, Ohio. Hadley, J.G., Conklin, A.M., Sanders, C.L. (1980) "Rapid solubilization and translocation of 109-Cd0 following pulmonary deposition". Toxicol. Appl. Pharmacol. 54:156-160.
748
G. OBERDORSTER, C. Cox and R. BAGGS
Holden, H. (1980) "Cadmium and pulmonary emphysema". Lancet 1:1137. lijima, Y., Takahashi, T., Fukushlma, T., Abe, S., llano, Y. and Kosaka, F. (1987) "Induction of metallothioneln by a macrophage factor and the partial characterization of the factor." Toxlcol. AppI. Pharmacol. 89:135-140. Kreyling, W.G., Bloom, S.D. and Brain, J.D. (1986) The uptake of soluble cobalt in the extrapulmonary airways of hamsters. In: Deposition and Clearance of Aerosols in the Human Respiratory Tract. Ed. W. Hoffmann, Facultas Universitatsverlag, Salzburg, pp. 62-77. Lundborg, M., Ling, B., Camner, P. (1984) "Ability of rabbit alveolar macrophages to dissolve metals". Exp. Lung Res. 7:11-22. Oberdorster, G., Baumert, H.P., Hochrainer, D., Stober, W. (1979) "The clearance of cadmium aerosols after inhalation exposure". Am. Ind. H~g. Assoc. J. 40:443-450. Oberdorster, kidney after Consultants. Environment.
G. and Kordel, W. (1981) "Metallothionein content in lung, liver, and chronic cadmium oxide and zinc oxide inhalation in rats." In: CEP Proceedings of the International Conference on Heavy Metals in the Edinburgh, pp. ~"0-~-5~5.
Post, C.T., Squibb, K.S., Fowler, B.A., Gardner, D.E., Illing, J., Hook, G.E.R. (1982) "Production of low molecular weight cadmium binding protein in rabbit lungs following exposure to cadmium chloride. Biochem. Pharmacol. 31:2969-2975. Takenaka, S., Oldiges, H., Konig, H., Hochrainer, H. and 0berdorster, G. (1983) "Carcinogenicity of cadmium chloride aerosols in Wistar rats. J. Natl. Cancer Inst. 70:367-373.
Thun, M.T., Schnorr, T.M., Smith, A.B., Halperin, W.E. and Lemen, R.A. (1985) "Mortality among a cohort of US cadmium production workers - An update". J. Natl. Cancer Inst. 74:325-333.