Lung deposition, lung clearance and renal accumulation of inhaled cadmium chloride and cadmium sulphide in rats

IOXICOIOGY E L S E V I E R S( I E N T I F I ( P U B L I S H E R S IRI- L A N D

Toxicology 84 (1993) 103-124

Lung deposition, lung clearance and renal accumulation of inhaled cadmium chloride and cadmium sulphide in rats H-J. Klimisch Department of Toxicology, BASF Aktiengesellschaft, D-6700 Ludwigshafen, Germany (Received 16 September 1992; accepted 18 May 1993)

Abstract Rats were exposed 6 h/day over 10 days to 0.3 mg/m 3 of water soluble cadmium chloride and 0.2, 1.0 and 8.0 mg/m 3 of insoluble cadmium sulphide, then killed at intervals over a 3month period for serial measurements of lung, renal and faecal cadmium. CdClz and highdose CdS animals showed a transient increase in lung weight. Clearance of both compounds was biphasic. Approximately 40% of deposited material was cleared during the 10-day exposure period. For CdCI2, only 9% of the lung burden was cleared rapidly after the last exposure (half-life 1.0 days) and 47% slowly (half-life 87 days), leaving a residual lung burden of 44%. For CdS, 41% of the lung burden was cleared rapidly (half-life 1.4 days) and 40% slowly (half-life 42 days), leaving a final residue 19%. In the CdS high-dose group, the retention of CdS in the lung was greater than that in the CdS low-dose groups, indicating that clearance mechanisms may possibly have been impaired in the high-dose group by too great a lung burden. For both compounds, faecal cadmium was initially high. Renal accumulation of cadmium was substantial for CdC12 during the exposure period and continued over the following months until it represented approximately 35% of the total cadmium cleared from the lung. For CdS, renal accumulation was only 1% of the amount cleared from the lung. The bioavailability of Cd from CdS is thus poor, the majority being cleared from the lungs and excreted in the faeces. However, the bioavailability of inhaled CdS measured as cadmium in the kidney is greater than the bioavailability of orally ingested CdS. Key words." Bioavailability; Cadmium chloride; Cadmium sulphide; Clearance; Lung;

I. Introduction Occupational cadmium exposure has been regulated on the basis of the cadmium content of materials rather than the actual toxicity of the compo0300-483X/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0300-483X(93)02627-S

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H-J. Klimisch / Toxicology 84 (1993) 103-124

nent compounds, but due to differences in aqueous solubility, the bioavailability and subsequent toxicity of cadmium compounds may vary considerably. In a study of soluble (CdCO3 and CdO fume) and insoluble (cadmium red and yellow sulphide pigments) cadmium compounds (Rusch et al., 1986) inhaled for 2 h by rats at a concentration of 100 mg/m 3 as cadmium, the soluble compounds caused lung damage and deaths with accumulation of cadmium in the liver and kidneys, while the insoluble compounds caused no deaths and cadmium was eliminated rapidly via the faeces with very little bioaccumulation. In the kidney, the organ with the longest biological half-life (10-30 years), only 0.07-0.1% of the inhaled CdS had been absorbed and accumulated in 30 days. The soluble cadmium chloride caused inflammatory lung lesions and hyperplasia of the bronchi, bronchioles and alveoli following 90 days exposure at concentrations of up to 50 /~g Cd/m 3 (Prigge, 1978). Cytotoxicity from cadmium chloride has also been shown to impair lung clearance (Dubreuil et al., 1979). Yet it has been suggested (Obersdoerster et al., 1979) that the bioavailability of cadmium from a variety of compounds may be independent of the chemical compound and its solubility. Cadmium sulphide did not show any carcinogenic potential after longterm inhalation in mice and Syrian hamsters (Heinrich et al., 1989), but the development of tumours of the respiratory tract has been reported in rats following inhalation of CdS (Oldiges et al., 1989). However, the significance of the latter finding has been questioned considerably since it was reported, the suggestion being that due to photodecomposition processes (Burlak et al., 1987; Ulicny and Gagliardy, 1990) substantial amounts of CdSO4 were formed from the CdS during the generation of the test atmosphere. In the study under question (Oldiges et al., 1989), a CdS aerosol was generated, not as a dry dust aerosol, but from an aqueous suspension of CdS in the presence of light through a jet atomizer and a glass cyclone and the same aqueous suspension was recycled through the system for many months. CdS crystals were being used due to their photoconductivity. However, visible light induces photoreduction of CdS and in the presence of water CdSO4 is formed (Burlak et al., 1987). This is soluble in water and should have a much greater bioavailability than the technically water-insoluble CdS. The aerosol generation method used in the original rat study (Oldiges et al., 1989), has recently been repeated in the same institute (K6nig et al., 1992) and the results have confirmed the formation of significant quantities of CdSO4. In another study (Glaser et al., 1986), the bioavailability of inhaled CdS in rats was reported to be very low, such that even CdO had a 10-times greater bioavailability than CdS. However, since the same basic generation technique was used as that in the previously mentioned long-term study (Oldiges et al., 1989), whereby a liquid suspension of CdS was nebulized with a jet

H-J. Klimisch / Toxicology 84 (1993) 103-124

105

atomizer, it is likely that even the reported low bioavailability data may result mainly from the formation of soluble CdSO 4 during the generation process rather than from CdS. The industrial applications of CdS are such that CdSO4 is not formed in normal use. In order to obtain information on possible differences in clearance, excretion and bioavailability of different cadmium compounds, the present study was performed with a water soluble compound (CdCI2) and a water insoluble compound (CdS). In this study, rats have been exposed by inhalation to cadmium chloride (generated as an aerosol from an aqueous solution) and to cadmium sulphide (generated as a dry dust to avoid problems of possible photoreduction) for 6 h/day for 10 days. Accumulation and clearance of cadmium from the lungs and renal cadmium accumulation have been determined from serial kills performed over a period from the second day of exposure up to 3 months after the exposure period, with daily measurement of faecal cadmium. Exposure concentrations for CdS were set at 8 mg/m 3 (equivalent to the 8 h limit value for inert dusts of the MAK list (1984)) and 0.2 mg/m 3 (3 times the MAK limit value for Cd), with an intermediate level of 1 mg/m 3. The cadmium chloride concentration was 0.3 mg/m 3. According to Prigge (1978), this concentration should cause tissue reactions in the lung and is almost equivalent to the lowest CdS concentration. 2. Materials and methods 2.1. Test materials The following test materials were used: cadmium chloride monohydrate (CdCIz.H20) (Merck, Darmstadt, Germany), 98% pure, containing 54.7% Cd, 34.5% C1, 8.8% H20 and 0.02% SO4, white crystalline powder and cadmium sulphide pigment (CdS) (cadmium yellow E), Europapigment, 98.2% pure, containing 75.9% Cd, 22.3% S, and 0.82% Zn, yellow crystalline powder (cadmium-soluble in 0.1 N HC1 (DIN 53 770) 0.051%). 2.2. Animals Three hundred male, specific pathogen free (SPF) Wistar rats (CHbb:THOM) (Thomae GmbH, Biberach, Germany), approximately 8 weeks old and weighing 250 g were used. The animals were housed individually in an air-conditioned room (temperature 20-24°C, relative humidity 30-70%) with a 12 h light/dark cycle and had free access to food ('Kliba' maintenance diet for rodents) and water (tap water) except during the inhalation exposures. 2.3. Exposure groups Five groups of 60 animals were exposed, nose-only, 6 h daily for up to 10

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H-J. Klimisch / Toxicology 84 (1993) 103-124

days (followed by a post-exposure observation period of up to 3 months) to the following test atmospheres: air control, 0.3 mg/m 3 CdCl2, 0.2, 1.0 and 8.0 mg/m 3 CdS. Four animals from each group were killed for deposition and clearance investigations on days 2, 10, 11, 12, 13, 17, 38, 66 and 94. 2.4. Inhalation exposure procedures The nose-only exposure chamber (Type INA 60 BASF AG) conditions were: airflow 100 l/min, mean temperature 23°C and mean relative humidity 49%. Cadmium chloride was generated as an aerosol by supplying an aqueous solution at a rate of 0.35 mg CdCIJmin from an injection pump to a binary nozzle atomiser (Mod. 970, Schlick) with compressed air. The aerosol was diluted with air and passed through a cyclone for the removal of large particles. Cadmium sulphide was generated as a dust in air at rates of 0.03, 0.16 and 1.46 mg/min by means of rotating brush-type generators (Technische Hochschule, Karlsruhe, Germany/BASF AG) and passed directly to the inhalation exposure chambers. Control animals were exposed to compressed air only. Cadmium chloride atmospheres were sampled at a velocity of 1.25 m/s through a 7 mm diameter sampling tube containing silica wool, 2 liquid impingers, a sintered-disk wash bottle (containing distilled water) a pump and an Elstner gas counter, and analysed using an Orion model 94-48 cadmium electrode. Cadmium sulphide atmospheres were also sampled at a linear velocity of 1.25 rn/s via a 7 mm diameter glass fibre filter and analysed gravimetrically. Particle size measurements were carried out twice during the study, using a cascade impactor (Andersen Mark III 6-stage stack sampler). Determinations were made gravimetrically except for Group 2 and the first sample from Group 3, which were analysed by atomic absorption spectrophotometry. 2.5. Cadmium deposition and clearance estimations For each interim kill, 4 rats were anaesthetized with Narcoren ® and exsanguinated. The lungs and kidney were removed and weighed. Organ samples were heated with sulphuric acid, evaporated with hydrogen peroxide and dissolved in 0.5 N HC1. Where the cadmium content was low, the cadmium was complexed with the ammonium salt of pyrrolidine-l-dithiocarboxylic acid and extracted with isobutyl methyl ketone (MIBK). Measurements were made using P-E model 305 and P-E model 4000 atomic absorption spectrophotometers (AAS) with a deuterium background compensator and an acetylene/air flame. Detection limits after complexation were: lung (1-2 g), 0.05 /zg Cd/g; kidney (1-2 g), 0.03 /zg/g. Faeces were collected daily throughout the study (including exposures) and pooled on a group basis. During the 10-day exposure period the faeces collected during exposures were analysed separately from the faeces collected from the holding cages.

H-J. Klimisch / Toxicology 84 (1993) 103-124

107

After drying, faeces were homogenized and 2-g samples were mineralized with sulphuric acid and hydrogen peroxide. The residue was evaporated twice with concentrated HC1 and taken up in 0.5 N HC1, then determined in MIBK by AAS (detection limit after complexation: 0.1 tzg Cd/g from a 2 g sample).

2.6. Statistical methods Body weight and lung weight data for days 10-'13 and for days 37, 65 and 93 were pooled and the means and standard deviations were calculated. Comparisons were made between Group 0 and Groups 2-4, using a t-test generalised by Williams (1971, 1972) for the simultaneous comparison of several dose groups with a control group. Groups 0 and 1 were compared using the method of Dunnett (1961,1964). Statistically significances at the 5% and 1% levels are indicated in Tables 3 and 5. The biological half-lives for the clearance of cadmium from the lungs following the last exposure were computed by the residual method (von Huttingberg et al., 1977). 3. Results

3.1. Exposure conditions Table 1 shows the mean chamber concentrations of cadmium chloride and cadmium sulphide over the 10 day exposure period. The data represent the fraction capable of being inhaled, i.e. collected in the sampling devices using a sampling velocity of 1.25 m/s. The mass median aerodynamic diameters (MMAD) 50% and slope factors for the samples taken from the atmospheres are shown in Table 2.

3.2. Clinical signs and body weights During the exposure and observation period there were no signs of adverse clinical findings or deaths. When the data for animals killed just after the

Table 1 Concentrations of cadmium compounds in test atmospheres (/~g/l air) Test group

Measuredmean

Target

Cadmium content

1 CdCI2 2 CdS 3 CdS 4 CdS

0.28 (0.08)a 0.22 (0.03) 0.92 (0.12) 8.08 (0.73)

0.3 0.2 1.0 8.0

0.17 0.17 0.72 6.29

aMean of 10 samples, figures in parentheses indicate S.D.

H-J. Klimisch / Toxicology 84 (1993) 103-124

108 Table 2 Particle size distribution of test atmospheres Test group

l

2 3 4

% alveolar deposition"

Sample 2

Sample 1 MMAD 50% t~m

Slope factor

MMAD 50% ~m

Slope factor

0.4 0.99 1.25 1.33

3.3 2.55 2.55 2.70

0.5 1.19 1.81 1.29

3.6 2.54 2.37 2.74

97 95 87 88

" % alveolar deposition: average % a veolar deposition based on MAK list definition of fine dust capable of alveolar deposition which is that fraction capable of passing through a trapping device capable of trapping 50% of particles having an aerodynamic diameter of 5 ~m. This represents stage 3 and subsequent stages of the cascade impactor.

exposure period (on days 10-13) are pooled (Table 3) there is a suppression of body weight gain relative to controls (P < 0.01) in the cadmium chloride group, but not in the cadmium sulphide groups. Pooling data for animals killed on days 38-94 showed no effect in any group.

3.3. Organ weights There were no effects on kidney weights. However, the absolute lung weights and lung weights relative to body weight of cadmium chloride-

Table 3 Comparison of body weights among groups killed (pooled data for days 10-13 and days 38, 66, 94)

Days 10-13 Mean S.D.

Group 0

Group l

Group 2

Group 3

Group 4

Control

CdC12 0.3 (t~g//l)

CdS 0.2 (t~g/l)

CdS 1.0 (~g/l)

CdS 8.0 (t~g/1)

281.10 14.25

265.40* 15.64

265.10 17.54

269.25 16.62

280.13 17.77

424.50 59.61

426.10 50.92

395.90 56.28

420.90 47.25

Days 37, 65, 93 Mean 413.40 S.D. 46.72

Means of 4 rats. *Significance, 1% in comparison with Group 0.

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H-J. Klimisch/ Toxicology 84 (1993) 103-124

exposed animals were found to be higher than controls (P < 0.01) for those killed over days 10-17, but not subsequently. This effect is shown in Table 4 in terms of pooled data for the sacrifices on days 10-13 and days 38, 66 and 94. There was a similar effect for the high dose cadmium sulphideexposed animals (P < 0.05) for pooled data from days 10 to 13.

3.4. Deposition and clearance The amounts of the test substances measured in the lungs of rats killed at intervals throughout the study are shown in Table 5 in terms of the mean amount per rat. Lung clearance is illustrated in Fig. 1 in terms of the percentage decrease in the lung concentration of cadmium in the 4 test groups. In all 4 test groups the clearance was biphasic and in each case can be described by two different first order exponential functions, the half-lives of which are shown in Table 6. 3.5. Renal accumulation of cadmium The cadmium content of the kidneys of rats killed at intervals throughout Table 4 Comparison of lung weights and lung weights relative to body weight among groups at sacrifice (pooled data for days 10-13 and days 38, 66, 94) Group 0

Group 1

Group 2

Group 3

Group 4

Control

CdC12 0.3 (#g/l)

CdS 0.2 (/~g/l)

CdS 1.0 (#g/I)

CdS 8.0 (/~g/1)

1.503 0.1413

1.744"* 0.1753

1.503 0.1413

1.416 0.1335

1.620" 0.1897

0.535 0.0613

0.657** 0.0588

0.536 0.0613

0.535 0.0516

0.578 0.0475

1.633 0.1353

1.653 0.2423

1.693 0.1786

1.628 0.1713

1.742 0.1754

0.397 0.335

0.392 0.0485

0.398 0.0192

0.414 0.0311

0.416 0.0419

Days 10-13

Weights Mean S.D. Ratio Mean S.D.

Days 37, 65, 93

Weights Mean S.D. Ratio Mean S.D.

Weights, absolute lung weights; Ratio, lung weight per 100 g body weight. Means of 4 rats. *Significance 5% compared with Group 0. **Significance 1% compared with Group 0

12.1 40.1 31.1 35.7 36.4 31.9 20.9 19.6 17.1

CdCI2 0.3 ~g/l

Control

(<0.06) (<0.07) (<0.07) (<0.08) (<0.08) (<0.07) (<0.08) (<0.08) (<0.12)

Group 1

Group 0

(7.4) (24.6) (19.1) (21.9) (22.3) (19.6) (12.8) (12.0) (10.5)

Cd +÷ (0.17 ~tg/1) 4.5 16.6 16.4 13.4 12.1 13.0 8.0 6.5 3.3

CdS 0.2 ttg/l

Group 2

Figures in parentheses represent cadmium content (ttg/rat).

2 10 11 12 13 17 38 66 94

Day

(3.5) (12.9) (12.7) (10.4) (11.8) (10.1) (6.2) (5.0) (2.6)

Cd ÷+ (0.17 /zg/l)

Table 5 Mean total lung cadmium chloride and cadmium sulphide (~g/rat)

15.0 46.6 42.3 46.4 44.3 39.0 19.9 15.5 10.6

CdS 1.0 ttg/l

Group 3

(11.6) (36.1) (32.8) (36.0) (34.3) (30.2) (15.4) (12.0) (8.2)

Cd ++ (0.72 ttg/l)

128.2 623.3 527.1 535.5 540.3 488.8 350.1 160.5 152.6

CdS 8.0 ~g/l

Group 4

(99.4) (483.2) (408.6) (415.1) (418.8) (378.9) (271.4) (124.4) (118.3)

Cd ++ (6.29 ttg/1)

I

t~

4~

2-

5~

H-J. Klimisch / Toxicology 84 (1993) 103-124

% lung

] 00

burden

90

Il I

k

80--

I,, '\\\

-

60--

50-40-30--

20-

]0-

O,

J

l

i

I0 ~

l

20

,

l

I

O3

,

,

40

l

50

,

l

,

60

,

70

,

l

l

80

,

I

90 Days

t

Ex )OSL/re period

Fig. 1. Percentage decrease in lung cadmium concentration after the end of the 10-day exposure period. • • , Group 1 (0.3 ~g CdCIJ1); o . . . . o, Group 2 (0.2 ~g CdS/1); ~ - - ~ Group 3 (1.0 ~g CdS/I); 4 - - --4, Group 4 (8.0 #g CdS/I). the study is s h o w n in Fig. 2 in terms o f m e a n c a d m i u m / g o f kidney. Total renal c a d m i u m t h r o u g h o u t the study is s h o w n in Table 7.

3.6. Faecal cadmium The c o n c e n t r a t i o n s o f c a d m i u m in the p o o l e d faeces f r o m each g r o u p recovered f r o m the h o l d i n g cages and e x p o s u r e cages are s h o w n in Table 8, Table 6 Half-lives of rapid and slow clearance phases of lung cadmium chloride and sulphide for each group Group l

Half-life (days) Rapid phase Slow phase

Group 2

Group 3

Group 4

CdC12 0.3 (t~g/l)

CdS 0.2 (t~g/l)

CdSI (k~g/l)

CdS 8 (~g/l)

1.0 86.0

1.4 39.8

2.9 51.0

1.2 35.9

112

H-J. Klimisch / Toxicology 84 (1993) 103-I24

~g Cd/g kidney A -- Group ~--~-....~Group ~m.,,,.~..m Group A,--~ ~ Group

3.0-

i 2 3 4

(0.3 ~g (0.2 ~g (i.0 ~g (8.0 ~g

CdCI2/I) CdS/l) CdS/l) CdS/I)

2.0

1.5

°'52 L

m,. 0 ~

0. i 0 ~ - ~ /

/

~g~"'~

U

~

o os- g/ . . . . . . 0.00

0

i0

20

30

40

50

60

70

80

90

I

Exposure period

Days

Fig. 2. Accumulation of cadmium in the kidney during and after the 10 day exposure period. • - • , Group 1 (0.3/zg CdC12/I); o - o, Group 2 (0.2/zg CdS/1); 13- - - 43, Group 3 (1.0 #g CdS/I); ~ zx, Group 4 (8.0 gg CdS/1).

Table 7 Cadmium deposition in kidneys (t~g Cd/rat) Day

2 10 11 12 13 17 38 66 94

Group 0

Group 1

Group 2

Group 3

Group 4

Control

CdCl 2 0.3 (gg/l)

CdS 0.2 (~tg/1)

CdS 1.0 (tzg/1)

CdS 8.0 (~zg/1)

0.06 0.06 0.06 0.07 0.06 0.07 0.12 0.07 0.14

0.10 2.06 1.91 2.32 2.58 3.16 3.74 6.21 7.04

0.06 0.06 0.06 0.06 0.06 0.07 0.08 0,12 0,31

0.06 0.07 0.06 0.06 0.07 0.07 0.14 0.25 0.34

0.06 0.52 0.48 0.5 I 0.71 0.93 1.90 2.53 4.18

H-J. Klimisch / Toxicology 84 (1993) 103-124

113

Table 8 Faecal cadmium (t~g Cd/rat) Day

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 24 31 45 59 73 87 94

Group 0

Group 1

Group 2

Group 3

Group 4

Control

CdCI 2 0.3 (#g/l)

CdS 0.2 (~g/1)

CdS 1.0 (#g/l)

CdS 8.0 (/~g/l)

1.84 2.13 1.99 1.76 2.24 2.74 0.77 2.25 2.97 2.38 1.71 2.62 2.75 2.75 2.75 2.10 3.22 . -. . . . --

1.41 6.05 4.40 3.60 5.16 5.29 4.44 4.20 3.56 4.45 4.23 3.40 2.92 2.92 2.92 5.87 3.13

1.13 8.61 14.27 11.26 3.13 a 8.14 10.77 13.00 13.49 20.83 10.43 3.93 3.72 3.72 3.72 3.55 6.31

.

0.94 3.68 7.88 5.48 15.54 11.15 6.58 6.60 3.90 4.66 10.26 2.57 5.17 5.17 5.17 3.49 2.48 .

. --

--

. . . .

. . . .

. . . . --

--

2.20 31.76 31.07 75.62 35.38 33.28 37.40 47.50 35.01 39.98 41.40 25.65 10.26 10.26 10.26 10.99 9.40 4.87 2.51 2.38 2.14 2.10 2.31 2.88

Measurements taken until day 17 for Groups 0-3 and continued until day 94 for Group 4. ~Sample from exposure cage lost.

4. Discussion

4.1. Lung deposition and clearance of cadmium chloride and cadmium sulphide during the exposure and post-exposure periods B a s e d u p o n t h e h i g h l y r e s p i r a b l e p a r t i c l e size d i s t r i b u t i o n s o f all f o u r t e s t a t m o s p h e r e s ( T a b l e 2), a h i g h e f f i c i e n c y o f l u n g d e p o s i t i o n is t o b e e x p e c t e d a n d b a s e d u p o n t h e a n a l y s e d a t m o s p h e r e c o n c e n t r a t i o n s , it is t o b e e x p e c t e d that the deposition would be close to the ratios of the nominal concentrat i o n s . A c o m p a r i s o n o f t h e l u n g C d S d e p o s i t s in t h e h i g h a n d i n t e r m e d i a t e d o s e g r o u p s a f t e r 2 d a y s o f e x p o s u r e ( T a b l e 5) s h o w s a p p r o x i m a t e l y 8 t i m e s as m u c h C d S in G r o u p 4 (128.2 tzg/lung) a s in G r o u p 3 (15.0 # g / l u n g ) , as ex-

114

H-J. Klimisch / Toxicology 84 (1993) 103-124

pected, but after 10 days the ratio of the CdS deposits has increased to 13.4. The CdS lung deposit in Group 4 is thus disproportionally high after 10 days, suggesting that lung clearance may be impaired due to the high lung CdS deposition. The lung deposit ratios between Groups 3 and 2 do not change in this way, although the lung deposits in Group 2 animals after both 2 and 10 days are somewhat higher than might be expected in comparison with those in Groups 3 and Group 4 (expected ratio between Group 3 and Group 2: 4.18/1, actual: 3.32/1). Such overload phenomena have been described previously for insoluble dusts starting at a lung deposit > 0.1 mg/g rat lung (Morrow, 1988; Pritchard, 1989; Muhle et al., 1990), but such an impairment of initial clearance may be compensated for in a later, postexposure phase as a result of an increased proliferation and stimulation of macrophages. Thus, in the present study, the clearance curves in the slow phase of clearance (Fig. 1) are fairly similar for all CdS groups. Such effects have also been reported after the inhalation of other insoluble particles such as carbon black or titanium dioxide (Muhle et al., 1990). Comparing Group 1 (CdCI2) with Group 2 (CdS), it is to be expected that the cadmium deposition in the lung would be approximately the same in both groups, since for both test atmospheres the analysed cadmium content was 0.17 #g/l. In practice, both after 2 and 10 days exposure, the cadmium content in the Group 1 animals (7.4 and 24.6/~g Cd/lung) was approximately twice that in the Group 2 animals (3.4 and 12.6/zg Cd/lung). This indicates either that the deposition of the liquid droplet CdC12 aerosol in the lung was more efficient that of the solid CdS dust, or that more CdC12 is retained in the lung, clearance being inhibited in some way (possibly due to the sequestration of ionic cadmium by metallothionein (Hart, 1986), or perhaps that both effects occur. When the maximum theoretical total deposition is calculated (Table 9), from the inhaled volume (based upon an assumed respiratory rate for the rat of 150 ml air/min taken from (Guyton, 1947) and a 6 h daily exposure duration), a mean of approximately 16% (14-21%) of theoretically available cadmium sulphide was retained in the lungs after 2 exposures and approximately double (38%) for cadmium chloride. This compares with results from other work of 13% deposition of CdCI2 reported after a 1-h exposure (Obersdoerster et al., 1979, 1980), 11% of 'cadmium fume particles' (Barrett et al., 1947) for up to 30 min and 12% after 5 exposures to CdO (Boisset et al., 1978). The clearance values of cadmium compounds from the lung during the post-exposure period are shown in Table 10, expressed in terms of /zg Cd/lung and as percentages of the lung deposits after 10 days exposure. The Table shows the amounts of cadmium after 10 days exposure and the residues after 94 days together with the amounts cleared rapidly and slowly, taken from the clearance curves. For CdS (Groups 2 and 3), approximately

108 (83.7) 15.0 (11.6) 13.9 46.6 (36.1) 38.4 (29.8) 45.2

22 (17.1) 4.5 (3.5) 20.5 16.6 (12.9) 9.0 (7.0) 35.2

12.1 (7.4) 37.8 40.1 (24.6) 31.1 (19.1) 43.7

CdS 1.0 ~, 0.72 b

32 (19.6) J

CdS 0.2", 0.17 b

CdCl 2 0.3 a, 0.17 b

Group 3

128.2 (99.4) 14.8 623.3 (483.2) 91.4 (70.9) 12.8

864 (670)

CdS 8.0 a, 6.29 b

Group 4

aConcentration #g Cd salt/l b Concentration p.g Cd++/l CCalculation formula: deposition = athmosphere concentration x exposure duration × respiratory minute volume. d#g Cd++/rat shown in parentheses.

Maximum theoretical deposition after 2 daily exposures c Residue after 2 exposures % of maximum theoretical Residue after 10 days Clearance during exposure period % cleared during exposure period

Lung cadmium compounds i~g/rat

Group 2

Group l

Table 9 Deposition and clearance (/zg cadmium compound/rat and #g Cd++/rat) of test materials in the lung during the 10 exposure days

I

4~

24.60

Cadmium chloride Group 1 2.28 (9.4%)

4.26 (32.6%) 15.88 (40.4%) 54.72 (12.8%)

Rapid clearance (~tg Cd/rat)

21.94 (90.6%)

8.81 (67.4%) 23.41 (59.6%) 374.08 (87.2%)

Remainder: slow or no clearance (>g Cd/rat)

"Measured residues at days 10 and 94 and amounts cleared taken from clearance curves.

12.90 36.12 483.18

Cadmium sulphide Group 2 ~ Group 3 Group 4

Residue at day 10 (~g Cd/rat)

11.44 (47.2)

6.21 (47.5%) 15.21 (38.7%) 255.78 (59.7)

Slow clearance (~g Cd/rat)

10.5 (43.4)

2.6 (19.9) 8.2 (20.9) 118.3 (27.6)

Residue at day 94 (>g Cd/rat)

Table 10 Rapid and slow phase lung clearance of cadmium and final residues during the post-exposure period, expressed in terms of #g Cd/rat and in terms of percentages of cadmium burden at day 10

I

{-

H-J. Klimisch / Toxicology 84 (1993) 103-124

117

37% of the lung burden after 10 days is cleared rapidly and approximately 44% during the slow phase, leaving a residue after 94 days of approximately 20%. For Group 4, the initial impairment of clearance due to overloading, which results in only 13% being cleared rapidly, is largely overcome during the slow clearance phase, so that after 94 days the residue is 28% of that after 10 days, compared to the 20% residue for the other 2 groups. There is, however, a clear difference between the clearance of CdC12 and CdS, in that despite its greater aqueous solubility, only 9.4% is cleared rapidly after 10 days exposure (compared to approximately 37% for CdS Groups 2 and 3) and the slow phase of clearance is also inhibited, so that approximately 43% of the total lung deposit of CdCI2 remains in the lung after 3 months, compared to approximately 20% of CdS (Table 10) and the half-life of the slow phase of clearance is approximately twice that for CdS (Table 6). It is therefore suggested that for CdC12, although a proportion of the material freshly deposited each day is cleared rapidly from the lung by tracheobronchial clearance or translocation, the remainder, possibly in the deep lung, is bound such that it can only be removed very slowly. This may be due to the cytotoxicity of CdC12 (Dubreuil et al., 1979), inhibiting mucocilliary clearance and phagocytosis, and/or due to inhibition of the transport of protein-bound CdCI2 via the lymphatic route (Obersdoerster et al., 1979). The increased lung weights in the CdC12 group (Table 2) are a sign of lung inflammation or oedema, which may be further evidence for a cytotoxic effect. This pattern of clearance is consistent with the observed rapid rise in renal cadmium during the exposure period and subsequent slower increase after exposure and is similar to results obtained in another study from a 1 h exposure to CdCI2 (Obersdoerster et al., 1979; Obersdoerster et al., 1980), in which half of the deposited cadmium was cleared during a 1.1 day half-life, with a 61 day half-life for the remainder. For CdS, clearance is rapid and efficient (retention time less than that for iron oxides (Obersdoerster et al., 1980)) and is probably due mainly to mucocilliary clearance, with secondary alveolar clearance of phagocytosed material carried onto the mucocilliary escalator or to the lymphatic system. These findings of differences in clearance and residual lung burden for CdC12 and CdS contradict assumptions in the literature (Obersdoerster et al., 1979) that cadmium compounds, irrespective of their chemical nature, form metal complexes in the lung and thus have similar half-lives, so that their toxic hazards would be similar. Our results would suggest that the toxic hazard associated with CdS would be considerably less than that of CdCI2, probably depending upon the degree of bioavailability or transformation into ionic cadmium. This is also supported by investigations (Rusch et al., 1986) showing an uptake of 6% after oral administration of water soluble, ionic cadmium and a much lower absorption of 0.07-0.1% after a single inhalation of water-insoluble, non-ionic CdS.

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H-J. Klimisch / Toxicology 84 (1993) 103-124

In considering the implications for extrapolation to humans of clearance data for 'insoluble' particles obtained from rats it is necessary to take account of the fact that rats have a relatively fast mechanical lung clearance whereas humans have a slower mechanical lung clearance of particles from the alveolar region with half-lives of a year or longer (Bailey et al., 1982; Bohning et al., 1982; Philipson et al., 1985). 4.2. Renal accumulation of cadmium Fig. 2 confirms that cadmium accumulates in the kidney after repeated inhalation of both a water-soluble cadmium compound (CdCI2) and an insoluble cadmium compound (CdS), for up to 90 days after exposure has ended, but that a much greater proportion of cadmium from CdCI2 accumulates in the kidney than from CdS, both during and after the exposure period. Table 11 shows the amounts of cadmium cleared from the lung after 10 days inhalation exposure and the amounts accumulated in the kidney. By the end of the exposure period, renal cadmium represents approximately 10.8% of cadmium cleared from the lung for CdC12, but less than 1% for all the CdS-exposed groups. The biphasic renal accumulation curve for CdC12 is steep during the exposure period (Fig. 2), after which accumulation is less rapid, but of the 14.1 #g Cd cleared from the lungs by the end of the post-exposure period, 35.3% (4.98 t~g Cd) reaches the kidney, which compares with 53% of cadmium appearing in the kidney following inhalation of CdO (Boisset et al., 1978). For CdS, there is a slow renal accumulation throughout the study, the final amounts representing 0.97-2.4'70 of the cadmium cleared from the lungs during the post-exposure period. 4.3. Faecal cadmium As Table 8 and Fig. 3 show, cadmium appeared in the faeces mainly during and for a few days after the exposure period. Table 12 shows the faecal cadmium during the first 11 days of the study compared with the lung clearance of cadmium during the 10 day exposure period (expressed in terms of t~g Cd/rat), For Groups 1-3 (CdCI2 and CdS), approximately 3 times as much cadmium was recovered from the faeces as was cleared from the lungs (Table 12), the added faecal burden probably representing cadmium compounds being deposited and rapidly cleared from the nasopharyngeal region (and therefore not appearing in the lung cadmium assays). Approximately twice as much cadmium was recovered from the faeces of the CdC12 group as from the equivalent CdS group (Group 2), which is consistent with the much greater initial lung deposition of cadmium in the CdC12 group. The ratio of faecal cadmium to cadmium cleared from the lung is lowest for the CdC12 group, supporting the finding that there is a greater systemic absorption of cadmium from C12 than from CdS. For Group 4 CdS, the faecal/lung cad-

2.4

35.3

Concentration #g Cd salt/1 b Concentration #g Cd++/1

0.31 0.25

7.04 4.98

Renal Cd day 94 (/~g/Cd/kidney) Renal Cd accumulation days 10-94 (#g Cd/kidney) Renal accumulation as % of lung clearance

2.6 10.3

10.5 14.1

Lung Cd day 94 (#g/Cd/lung) Lung Cd clearance day 10-94 (/xg/Cd/lung)

12.9 6.98 0.06 0.9

24.6 19.08 2.06 10.8

CdS 0.24, 0.17 b

CdCI 2 0.3 ", 0.17 b

Lung Cd day 10 (#g/Cd/lung) Lung Cd clearance to day 10 (/zg/Cd/lung) Renal Cd day 10 (~g/Cd/kidney) % of cleared cadmium in kidney

Group 2

Group 1

0.97

0.34 0.27

8.2 27.9

36.1 29.77 0.07 0.2

CdS 1.0~, 0.72 b

Group 3

1.00

4.18 3.66

118.3 364.9

483.2 70.85 0.52 0.7

CdS 8.0 a, 6.29 b

Group 4

Table 11 Comparisons of renal accumulation of cadmium during exposure and post-exposure periods with lung cadmium clearance over the same periods

I

oo 4~

H-J. Klimisch / Toxicology 84 (1993) 103-124

120

Faeces

Kidney

Fg

Lung ~g

Cd/g

15

e faeces m-------m l u n g -- --~ k i d n e y

1 .5

/,,A

Cd/g

3OO

/ /

I%\ \ I 10

/ ,/

\

l

1.0

25O

,/

\

s,

./

\

/

W

20O

/

]5O

5

\

0.5

1 O0

5O

0

0.C

!

I

110

0 I

I

I

20 I

I

I

30

i

i

40

i

!

50

i

!

60

!

i

70

i

!

80

Exposure period

i

i

0

90

Days

Fig. 3. Lung, kidney and faecal cadmium in group 4 (8 mg CdS/1) during and after the 10-day exposure period; • 0, faeces; ~ ---l lung; A A, kidney. mium ratio is the highest (5.5:1), which again illustrates the impaired clearance from the overloaded lungs of the high-dose CdS group. These data also confirm the finding that the most prominent elimination route of CdS after oral administration, or of cadmium in the gastrointestinal tract after clearance from the upper respiratory tract, is excretion via the faeces (Rusch et al., 1986; Loeser, 1974); only very small amounts being absorbed systemically and accumulating in the kidney.

4.4. Bioavailability of cadmium chloride and cadmium sulphide From the differences in the clearance and renal accumulation of CdC12 and CdS it has been shown that the bioavailability of insoluble cadmium compounds is much less than that of soluble compounds, confirming the findings of other authors following intake orally and by inhalation (Rusch et al., 1986; Loeser, 1974). The bioavailability of cadmium is thus dependent on the chemical nature of cadmium compounds and their solubility (Obersdoerster et al., 1979). It is not clear to what extent inhaled cadmium compounds are bioavailable directly from the lungs or from the gastrointestinal tract. For CdCIz the rapid renal accumulation and relatively low faecal recovery of cadmium during the exposure period may represent solubi-

total faecal Cd during first 11 days of study (/~g Cd/rat), minus control background levels Lung Cd clearance to day 10 (p,g/Cd/lung) Ratio of faecal Cd to lung clearance

-

24.0

6.98 3.4

19.08 2.8

CdS 0.20zg/1)

CdCI 2 0.3 (txg/1) 53.9

Group 2

Group 1

29.77 3.1

92.3

CdS 1.0 (t~g/1)

Group 3

Table 12 Comparisons of faecal cadmium with cadmium clearance from the lung during the exposure period

70.85 5.5

387.8

CdS 8.00zg/1)

Group 4

4~

I

4~

g.

122

H-J. Klimisch / Toxicology 84 (1993) 103-124

lization and absorption or translocation of cadmium, either directly from the lung or by gastrointestinal absorption of cadmium compounds cleared from the lung and swallowed. Similarly, for CdS there is the question of whether cadmium undergoes biotransfer (by resorption or active biotransformation) from the lung to accumulate in the kidney, or whether clearance and subsequent absorption from the gastrointestinal tract are solely responsible. Orally administered CdS results in a very low renal accumulation of cadmium (Loeser, 1974; Mangler et al., 1983) and this may also be the route of absorption for inhaled and cleared CdS. It is possible to calculate a resorption factor (%) showing how much orally ingested CdS has accumulated in the kidneys (Loeser, 1974). The amount of CdS absorbed from the gut is calculated from the amount of CdS in the 24 h faeces and the oral dose. This resorption factor of 0.02% after 28 days of oral application is very low, considering that cadmium pigments (CdS) contain cadmium soluble to 0.05% in 0.1 M HC1. After a 2 h inhalation exposure followed by a post-exposure observation period of 30 days (Rusch et al., 1986), the resorption factor is 0.07-0.1%. The supplied amount of CdS is again calculated from the total faecal amount over 30 days. Thus, the resorption factor after a single inhalation, including a 30 day post-exposure period, is about 5 times higher that after continuous oral application over 28 days. In our inhalation study, resorption factors were also higher than those obtained after oral ingestion. The resorption factor even increased further during in the post-exposure period (Table 13). The inference is therefore, that renal cadmium accumulates at least to some extent from CdS translocated from the lung, rather than from the gut. It is known that the lung content of metallothionein, which may be an important factor in the bioavailability process of non-ionic cadmium, increases after repeated exposures of insoluble CdS probably in relation to a long-lasting lung burden of CdS (Hart, 1986). This would correlate with the increasing resorption factor. In addition, it has to be considered that after approximately day 44, the concentrations of cadmium in the faeces in the highest CdS concentration group correspond to those in the control animals, so that the gradual renal accumulation shown in Fig. 3 most likely results from the slow bioavailability

Table 13 Resorption factors (%) of orally ingested CdS accumulated in the kidneys Test days

Resorption factor (%)

10 16 37

0.12 0.19 0.29

H-J. Klimisch / Toxicology 84 (1993) 103-124

123

o f residual lung c a d m i u m deposits. It is k n o w n that alveolar m a c r o p h a g e s can dissolve particles generally r e g a r d e d as 'insoluble' ( L u n d b o r g et al., 1992). Alternatively, after mucocilliary clearance and a b s o r p t i o n f r o m the gastrointestinal tract, c a d m i u m m a y be deposited in a n o t h e r organ, such as the liver (Rusch et al., 1986; D o r i a n et al., 1991), released as metallothioneincomplex into the circulation and t a k e n up by the kidney. H o w e v e r , as r e p o r t e d by H a r t (1986), p r i o r or repeated c a d m i u m - i n h a l a t i o n exposure increased c a d m i u m t r a n s l o c a t i o n to the kidney, but not to the liver. T a k i n g all aspects into consideration, it can be c o n c l u d e d that a s o m e w h a t higher bioavailability o f inhaled CdS c o m p a r e d with ingested CdS c a n n o t be ruled out. H o w e v e r , overall, the bioavailability o f CdS is substantially lower than that o f soluble c a d m i u m c o m p o u n d s . 5. Acknowledgment The a u t h o r is very grateful to D.A. P u r s e r for his excellent help in writing a m a n u s c r i p t o f this w o r k for publication.

6. References Bailey, M.R., Fry, F.A. and James, A.C. (1982) The long-term clearance kinetics of insoluble particles from the human lung. Ann. Occup. Hyg. 26, 273. Barrett, H.M., Irwin, D.A. and Semmons, E. (1947) Studies on the toxicity of inhaled cadmium. J. Ind. Hyg. Toxicol. 29, 279. Bohning, D.E., Atkins, H.1. and Cohn, S.H. (1982) Long-term particle clearance in man: normal and impaired. Ann. Occup. Hyg. 26, 259. Boisset, M., Girard, F., Godin, J. and Boudene, C. (1978) Cadmium content of lung, liver and kidney in rats exposed to cadmium oxide fumes. Int. Arch. Occup. Environ. Health 41, 41. Burlak, A.V., Ignatov, A.V., Serdynk, V.V. and Furlei, A.J. (1987) Mechanism of degradation of the photoconductivity of cadmium sufide single crystals. Sov. Phys. Semicond. 21, 1035. Deutsche Forschungsgemeinschaft, MAK List: 'Maximale Arbeitsplatzkonzentratioen (MAK), Biologische Arbeitsstofftoleranzwerte (1984) Mitteilung XX der Senatskommission zur Priifung gesundheitsschfidlicher Arbeitsstoffe, Verlag Chemic, Weinheim. ISSN 0417-1810; 3-527-27553-3. Dorian, C., Gattone, V.H. II and Klassen C.D., (1991) Studies on the mechanism of cadmium nephrotoxy. Toxicologist 11, Ref. 871, p. 231. Dubreuil, A., Bouley, G. and Boudere, C. (1979) In vitro cytotoxicity of cadmium microparticles for rabbit alveolar macrophages. Scand. J. Work Environ. Health 5, 211. Dunnet, C.W. (1961) Multiple comparison between treatments and a control. Biometrics 17, 324. Dunnet, C.W. (1964) New tables for multiple comparisons with a control. Biometrics 20, 482. Glaser, U., Kloppel, H. and Hochrainer, D. (1986) Bioavailability indicators of inhaled cadmium compounds. Ecotoxicol. Environ. Safety 11,261. Guyton, A.C. (1947) Analysis of respiratory patterns in laboratory animals. Am. J. Physiol. 150, 78.

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H-J. Klimisch / Toxicoh~gy 84 (1993) 103-124

Hart, B.A. (1986) Cellular and biochemical response of the rat lung to repeated inhalation of cadmium. Toxicol. Appl. Pharmacol. 82, 281. Heinrich, U., Peters, L., Ernst, H., Rittinghausen, S., Dasenbrock, C. and K6nig, H. (1989) Investigation on the carcinogenic effects of various cadmium compounds after inhalation exposure in hamsters and mice. Exp. Pathol. 37, 253. K6nig, H.P., Heinrich, U., Kock, H. and Peters, L. (1992) Effect of photocorrosion on cadmium sulfide suspensions applied in animal inhalation studies with CdS particles. Arch. Environ. Contain. Toxicol. 22, 30. Loeser., E. (1974) Cadmium chloride, cadmium pigment yellow, cadmium pigment red: subchronic toxicological investigations on rats (3-month feeding trial). Bayer AG: Report No. 4639. BAYER AG, Friedrich-Ebert-Str. 217, D-5600 Wuppertal 1. (Unpublished.) Lundborg, M., Falk, R., J ohansson, A. (1992) Phagosomal pH and dissolution of cobalt oxide particles by alveolar macrophages. Environ. Health Perspect. 97, 153. Mangler, B., Haberle, K., Fischer, G. and Classen. H.G. (1983) Enteral absorption and retention of cadmium in rats fed Cd as the sulfide and chloride at two levels during 90 days. Naunyn-Schmiedeberg's Arch. Pharmacol. 322 Suppl, R125 (Abstract). Morrow, P.E. (1988) Possible mechanism to explain dust overloading of the lungs. Fundam. Appl. Toxicol. 10, 369. Muhle, H., Bellmann, B., Creutzenberg, O., Bellmann, B., Heinrich, U., Ketkar, M. and Mermelstein, M.R. (1990) Dust overloading of lungs after exposure of rats to particles of low solubility. Comparative studies. J. Aerosol Sci. 21, 374. Muhle, H., Creutzenberg, O., Bellmann, B., Heinrich, U. and Mermelstein, R. (1990) Dust overloading of lungs: investigations of various materials, species difference and irreversibility of effects. J. Aerosol Med. 3, 64. Obersdoerster, G., Baumert, H.-P., Hochrainer, D. and Stoeber, W. (1979) The clearance of cadmium aerosols after inhalation exposure. Am. Ind. Hyg. Assoc. J. 40, 443. Obersdoerster, G., Oldiges, H. and Zimmermann, B. (1980) Lung deposition and clearance of cadmium in rats exposed by inhalation or by intratracheal instillation. Zentralbl. Bakteriol., 1. Abt. Orig. B 170, 35. Oldiges, H., Hochrainer, D. and Glaser, U. (1989) Long-term inhalation study with wistar rats and four cadmium compounds. Toxicol. Environ. Chem. 19, 217. Philipson, K., Falk, R. and Camner, P. (1985) Long-term lung clearance in humans studied with Teflon particles labelled with chromium-51. Exp. Lung Res. 9, 31. Prigge, E. (1978) Early signs of oral and inhalative cadmium uptake in rats. Arch. Toxicol. 40, 231. Pritchard, J.N. (1989) Dust overloading causes impairment of pulmonary clearance: evidence from rats and humans. Exp. Pathol. 37, 39. Rusch, G.M., O'Grodnick, J.S. and Reinhart, W.E. (1986) Acute inhalation study in the rat of comparative uptake, distribution and excretion for different cadmium containing materials. Am. Ind. Hyg. Assoc. J. 47, 754. Ulicny, J. and Gagliardy, G.B. (1990) Photodecomposition of CdS. Chemistry Report Cadmium Sulfide Pigment, 18 September. SCM-CHEMICALS, 2701 Brolming Highway, Baltimore, MD 21222, USA (Unpublished.) Von Huttingberg, H.P., Brockmeier, D. and Krouter, G. (1977) A rotating iterative procedure (RIP) for estimating hybrid constants in multi-compartment analysis on desk computers. Eur. J. Clin. Pharmacol. 11, 381. Williams, D.A. (1971) A test for difference between treatment means when several dose levels are compared with a zero dose control. Biometrics 27, 103. Williams, D.A. (1972) The comparison of several dose levels with a zero dose control. Biometrics 28, 519.