N rats exposed for 1–22 days

Toxicology 103 (1999) 9-22

Pulmonary toxicity of nickel subsulfide in F344/N rats exposed for l-22 days ’ Janet M. Benson*, Yung-Sung Cheng, Arthur F. Eidson ‘, Fletcher F. Hahn, Rogene F. Henderson, John A. Pickrel12 Inhahtion Toxicology Research Institute, P.O. Box 5890. Albuquerque, NM 87185. USA

Received 30 December 1994;accepted 13 March 1995

Repeated inhalation of nickel subsulfide (Ni&) by F344M rats for 3 months results in chronic active inflammation in the lung and atrophy of the olfactory epithelium. The primary purpose of this study was to determine early responses of the respiratory tract to inhaled NisS, in rats and to track the course of development of such lesions in rats exposed for up to 22 days. A secondary purpose was to obtain an improved estimate of the half-time for clearance of Ni from Ni&exposed lungs. Groups of F344iN rats were exposed to 0, 0.6 or 2.5 mg Ni&m3, 6 h/day for l-22 days. Histopathological changes in nose and lung, as well as biochemical and cytological changes in lung, as measured in bronchoalveolar lavage fluid (BALF) and lung tissue, alveolar macrophage (AM) viability and Ni concentration in lung were evaluated. Inflammatory lung lesions in rats exposed to 2.5 mg Ni3Sr/m3 peaked in intensity after 4 days of exposure. Minimal degeneration of the olfactory epithelium was noted in the 2.5 mg Ni3S2/m3-exposed rats after day 4 of exposure, with atrophy of the olfactory epithelium occurring in rats killed at 22 days. Lactate dehydrogenase, &glucuronidase and total protein in BALF were significantly elevated within 7 days of exposure while alkaline phosphatase activity was significantly depressed. AM viability was significantly reduced after 2 days of exposure. Concentrations of Ni in lung increased rapidly during the first 7 days of ,exposure, but more slowly thereafter. Lung burden data from this and a previous study suggest a clearance half-time for Ni of 3.5-8 days. Results indicate that Ni,S, is relatively soluble in lung and inhalation of concentrations near the current Threshold Limit Value of 1 mg Ni/m3 can produce detrimental changes in the respiratory tract of rats after only a few days of exposure. Keywords: Nickel subsulfide; Inhalation;

Rat; Histopathology;

Bronchoalveolar

lavage fluid

* The submitted ntanuacript has been authored by a contractor, Lovelace Biomedical and Environmental Research Institute, of the U.S. Government under Deparbnent of Energy Contract Number DE-ACW76EV01013. Accordingly, the U.S. Government retains nonexclusive royalty-free license to publish or reproduce the published form of this contribution or allow others to do so, for U.S. Govemmentpumoses. l Correspcaxiingauthor. ’ Current Addressz GntemationaITechnology Corporation, Albuquerque, NM 87108, USA. 2 Current Address: Colkge of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA. 0300-483X.B5BO9.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0300-483X(95)03098-Z

10

J.M. Benson et al. / Toxicology 103 (I995)

1. Introduction Excess risk of nasal and lung cancers has been reported among individuals employed in the nickel (Ni) refining industry during the earlier part of this century (Report of the International Committee on Nickel Carcinogenesis in Man, 1990). Epidemiological data indicated that excess risk of lung and nasal cancers was associated with exposure to dusts containing nickel subsulfide (Ni&) and other forms of Ni. Inhalation of Ni does not appear to be associated with excess risk of developing pneumoconiosis or chronic respiratory disease (Mastromatteo, 1990). Ottolenghi et al. (1974) have shown that Ni& is also a pulmonary carcinogen in F344/N rats. In their studies, rats were exposed to approximately 1 mg Ni3S2/m3 (0.73 mg Ni/m3) 6 h/day, 5 days/ week for 78 weeks. This exposure regimen resulted in a 14% incidence in lung tumors, compared to 1% in controls. The incidence of inflammatory lesions was also increased in the Ni&-exposed rats. More recently, groups of F344/N rats were exposed by inhalation to 0.15-2.5 mg Ni3S,/m3 (0.11-1.8 mg Ni/m3) 6 h/day, 5 days/week for 13 weeks (Dunnick et al., 1989; Benson et al., 1990). Pulmonary lesions included alveolar macrophage (AM) hyperplasia (at 0.15 mg Ni3S2 and above) chronic active inflammation (at 0.15 mg/m3 and above), mononuclear cell infiltration of the interstitium (at 0.3 mg/m3 and above) and hyperplasia of the bronchiolar epithelium (at 0.6 mg Ni3S2/m3 and above). Atrophy of the nasal olfactory epithelium generally occurred in rats exposed to ~0.6 mg Ni3Sz/m3. The incidence and severity of the pulmonary and nasal lesions increased with N&S2 exposure concentration. The morphological changes in lung occurring in rats exposed to 0.6 and 2.5 mg Ni3S2/m3 have been associated with statistically significant increases in biological markers of cytotoxicity and inflammation in bronchoalveolar lavage fluid (BALF) (Benson et al., 1989a). Equilibrium concentrations of Ni in lungs of the Ni3S2 rats appeared to have been reached during the 13-week exposure period (Dunnick et al., 1989). The inflammatory changes occurring in Ni3S2-exposed F344/N rats have not been documented in humans. However, the changes

9-22

may be important to the development of neoplasms in rats (and humans) chronically exposed to this compound and, therefore, warrant evaluation. Several unanswered questions regarding the inhalation toxicity of Ni3S2 remain to be addressed. First, how soon do respiratory tract lesions occur among rats inhaling occupationally relevant aerosol concentrations of Ni,S,? Second, when during a 13-week exposure period to Ni& are equilibrium concentrations of Ni in lungs reached? Such information may be useful in predicting the health risks to humans associated with exposure to Ni sulfides. The major objective of this study was to determine the time- course of morphological and biochemical changes in the lungs of F344/N rats exposed by inhalation to 0, 0.6 or 2.5 mg Ni3S2/ m3 for 1, 2, 4, 7, 12 or 22 days. A secondary objective was to obtain an estimate of the half-time for clearance of Ni from the lung by clearly identifying the time at which equilibrium Ni concentrations are reached in lungs of Ni&-exposed rats. The Ni3S2 aerosol concentrations chosen were expected to produce an inflammatory reaction in lung over the course of the 22-day exposure. 2. Methods 2. I. Chemical Nickel subsulfide (beta-Ni,S,, CAS No. 1203572-2) was supplied by the National Toxicology Program, National Institutes of Environmental Health Sciences (Research Triangle Park, NC) through the Midwest Research Institute (MRI, Kansas City, MO). Chemical analysis conducted by the MRI indicated the material was 97% pure. 2.2. Exposure system Rats were exposed whole body to target aerosol concentrations of 0 (control), 0.6 and 2.5 mg Ni3S,/m3. Aerosols were generated using 2-in stainless steel fluidized bed aerosol generators containing type 316 stainless steel powder (Benson et al., 1990). A generator containing bed material without added Ni& was used to supply the control exposure chamber. The Ni& aerosol concentrations were quantitated by taking filter samples

J.M. Bensonet at./ Toxicology103 (1995) 9-22

(Zefluor, 1 pm pore, Gelman, Ann Arbor, MI) for three, 2-h periods during the 6-h exposure day at a flow rate of 3 l/mm. The aerosol mass concentration was calculated from the mass of material collected on the filter, sampling flow rate and sampling time. An aerosol mass monitor (RAM-S, MIE Corporation, Bedford, MA) was operated for at least 5 min ad every exposure hour in each Ni& chamber to obtain real-time information on the stability of the aerosol concentration. The mass median aerodynamic diameter and geometric standard deviation were determined at each exposure concentration using Lovelace multijet cascade impactors.

11

3. Experimental design 3.1. General The experimental design is summarized in Table 1. Briefly, groups of 90- 132 F344M rats were exposed 6 h/day to target concentrations of 0,0.6 or 2.5 mg Ni&/m3, for up to 22 days. Subgroups of rats, as indicated in Table 1, were sacrificed by exsanguination from the axillary arteries while under halothane anesthesia (4% in 02) at designated intervals during the study. 3.2. Quantitation of Ni in lung After sacrifice, lungs were removed from the rats using surgical instruments with tungsten carbide contact surface inserts to minimize Ni contamination. The lungs were weighed and stored at -20°C in plastic vials until analyzed. Tissues were digested in an acid mixture containing 1 ml 16 M HNO,, 0.5 ml 6 M HCl and 1 ml 30% Hz02 per g of tissue in teflon digesters using a microwave oven, The Ni contents of the digests were determined using electrothermal atomic absorption spectroscopy. The details of the digestion and analysis procedures have been reported (Benson et al., 1989b). The limits of detection (LOD) and

2.3. Animals Equal numbers of male and female F344M rats born and raised at the Institute were used. The rats were acclimatized for 19 days in Hazleton 2000 multitiered inhalation chambers (Lab Products, Maywood, NJ) before exposures began. The rats were randomized. by weight into experimental groups and were approximately 7 weeks old at the start of exposures. Food (NIH-07 Certified diet, Ziegler Brothers, inc., Gardners, PA) was provided during nonexposure hours. Water was provided ad libitum.

Table 1 Experimental design Sacrifice time (days of exposure)

Rats per sacri!icep (mg Ni$dm3)

Lung burden

Histopathology

B&hemistry

Lung burden

1 2 4 I 12 22

6 0 6 0 0 -6

6 0 0 0 0 -6

10 10 10 10 10 10 -

6 6 6 6 6 -6

Subtotal

18

12

60

36

Total

2.5

0.6

0

90

Biochemistxy

Lung burden

6 0 0 6 0 -6

10 10 10 10 10 -IO

6 6 6 6 6 -6

6 6 6 6 6 -6

10 10 IO 10 10 -10

18

60

36

36

60

114

V?qual numbers of males and females comprise each group.

Histopathology

Biochemistry

Histopathology

132

12

J. M. Benson et al. / Toxicology 103 (1995) 9-22

quantitation (LOQ) of the method were calculated as follows (Keith et al., 1983): LOD = 6 + 3 SDt, and LOQ = 6 + 10 SDt, where 6 is the cumulative mean of value for Ni obtained for the acid digest matrix ‘blank,’ and SDb is the cumulative standard deviation of the mean ‘blank’ values. Concentrations of Ni in lung are expressed in pg Ni/lung and as rg Ni/g control lung. In the latter case, control animal lung weights were used to normalize any increase in lung weight due to pathological changes and to allow combining of lung burden data from male and female rats. 3.3. Biochemical and cytological evaluation of lavage fluid

The following were quantitated in BALF: lactate dehydrogenase (LDH), an indicator of cell death or cell membrane damage; total protein (TP), an indicator of increased permeability of the alveolar/capillary barrier; /3-glucuronidase (BG), an indicator of increased activity or lysis of macrophages; alkaline phosphatase (AP), an indicator of type II epithelial cell secretion or damage; collagenous peptides (CPs), an indicator of lung collagen turnover; and glutathione (GSH), a polypeptide thought to protect against oxidant damage. GSH and CPs were also quantitated in tissue homogenates. Activities of LDH, BG and AP and the concentration of TP in BALF were determined according to previously described procedures (Henderson et al., 1979; Benson et al., 1989a). Enzyme activities are expressed in terms of mIU/g control lung, where an IU is defined as pmol of substrate hydrolyzed per min at 37°C. CPs in BALF and lung tissue homogenates were determined as hydroxyproline according to the method of Grant (1965). Concentrations of CPs in lung were obtained by multiplying corresponding concentrations of hydroxyproline by 7.46 (Neuman and Logan, 1950). GSH concentrations in lung lavage fluid and tissue homogenates were determined according to Sun et al. (1988) using a modification of the method of Cohn and Lyle (1966). Lung lobes were

homogenized in 20 mM EDTA/SO mM Tris-HCl buffer. To 400 ~1 of homogenate, 100 pl of 25% phosphoric acid was added to precipitate proteins, which were sedimented by centrifugation at 3000 x g for 5 min. The supernatant fraction was neutralized by addition of 180 ~1NaOH. The samples were diluted to 1 ml with 20 mM EDTA and each sample was reacted with 0.1% o-phthalaldehyde (in methanol) at room temperature for 20 min. The fluorescence from each sample was measured at an excitation wavelength of 350 nm and an emission wavelength of 420 nm. Standard curves were prepared using GSH. Total nucleated cells (TNCs) recovered in lavage fluid and differential cell counts were determined as previously described (Benson et al., 1989a). The viability of the AM was determined by their ability to exclude the dye trypan blue. 3.4. Morphological changes Groups of rats were sacrificed as indicated in Table 1 for evaluation of morphological changes occurring in lung and nasal tissue. The lungs, trachea, larynx and nasal tissues (head with skin, mandible and brain removed) were removed, examined grossly and placed in Karnovsky’s tixative. The lungs were perfused through the trachea at a pressure of 25 cm Hz0 for 24 h. Tissues were imbedded in parafftn, cut at 5 pm and stained with hematoxylin and eosin. The lesions were qualitatively scored from 1 (minimal lesion ) to 4 (severe lesion) based on their intensity and distribution. 3.5. Statistical evaluation Because the biochemical, cytologic and morphological changes observed among male and females were qualitatively and quantitatively the same, data for males and females were combined for statistical evaluation. Lung burden data for males and females have also been combined. Ni concentrations in lung and biochemical parameters are expressed in terms of units/g control lung to normalize for increases in lung weight in the Ni,&exposed groups due to pathological changes. The significance of differences between Niexposed and sham-exposed (control) group means was assessed by analysis of variance adjusted for multiple comparisons (BMDP software). Differ-

J.M. Lknson CI aI. / Toxicology IO3 (1995) 9-22

13

ences at a significance level of P 5 0.05 are reported. 4. Rem&s 4.1. General Aerosol concentrations (mean f S.E.M.) achieved over the 22-day exposure period were 0.59 f 0.03 mg Ni3Sz/m3 and 2.5 f 0.03 mg Ni&/m3. The mass median aerodynamic diameters (geometric standard deviations) for the 0.6 and 2.5 mg Ni3S2/m3 aerosols were 2.07 pm (2.15) and 1.98 pm (2.03). Inhalation of 0.6 mg N&/m3 had no effect on weight gain of male and female rats on study. However, the body weights of male rats exposed to 2.5 mg Ni3Sz/m” were significantly lower than those of control males after 7 days of exposure, while weights of female rats were significantly lower than control females only after 7 and 12 days of exposure (Fig, IA, B). A significant drop in body weights of male and females exposed to 2.5 mg Ni3Sz/m3 was noted in rats sacrificed after day 7 of exposure. The rats in this exposure group appeared lethargic at that time and one male and one female rat died during day 7 of exposure. This effect could not be correlated with a large deviation of aerosol concentration from target values or with adverse environmental conditions. Clinical signs of toxicity resolved after this point and no

I-_.-0

200

6

18 12 DAYS OF EXPOSURE

B

Females

1

‘40 -

0

, 0

I 6

I 18

I 12 DAYS

Fig. 1. Body weights of control and Ni&-exposed male (A) and female (B) rats. Points represent the mean f S.E.M. of five values.

Days of exposure

CXpOSUrC

Broup (mdm?

2

4

I

12

22

0.07 0.03 0.03

0.94 l 0.02 1.03 l 0.02 1.08 l 0.04

1.06 f 0.06 1.04 l 0.06 1.45 f 0.04b

1.03 f 0.06 1.22 * O.OSb 1.45 f 0.09b

1.19 * 0.09 1.31 l 0.04b 1.58 zt 0.07b

1.16 f 0.17 1.37 f 0.07b 2.23 * 0.08b

0.05 0.04 0.03

0.85 f 0.05 0.95 f 0.04 1.02 * 0.02

0.89 f 0.04 0.90 t 0.04 1.16 + 0.06b

0.92 f 0.07 1.05 f 0.03 1.29 LIZ 0.14b

0.95 f 0.05 1.25 l 0.04b 1.35 l 0.02b

1.02 f 0.05 1.30 f 0.13b 1.69 l 0.03b

I

Male lung weight (g) 0.93 :t 0 1.02 :t 0.6 0.98 i 2.5 Female lwrg weigh: (g) 0.81 :t 0 0.85 :t 0.6 0.85 it 2.5

I 24

OF EXPOSURE

Table 2 Effect of Ni& exposure on lung weight* N&S2

24

*Results represent thle mean f S.E.M. of five values. bMean signiticantly different from control; analysis of variance adjusted for multiple comparisons. Significance level P S 0.05.

J.M. Een.~n et al. /Toxicology

14

additional mortality occurred. At the end of the 22-day exposure period, body weights of males exposed to 0.6 and 2.5 mg Ni&/m3 were 85% and 82% of control male body weights, respectively. Females in the 0.6 and 2.5 mg Ni3S2/m3 groups weighed 93% and 89% of control females, respectively. Lung weights of rats in the 0.6 and 2.5 mg Ni3S2/m3 groups were significantly increased by days 12 and 4 of exposure respectively (Table 2) and remained elevated thereafter. 4.2. Nickel lung burdens Quantities of Ni in control lungs were below the limit of detection of the method (0.660 pg Ni/lung) at all sacrifice times (data not shown). Quantities of Ni in lungs of rats exposed to 0.6 mg Ni3S2/m3 for 1 day were below the limit of quantitation (1.87 pg Ni/lung). Concentrations of Ni in lungs of males and females were similar; therefore, data from males and females were pooled. Concentrations of Ni in lungs of rats exposed to 0.6 mg Ni&/m3 increased from 3.0 to 12 pg Ni/g control lung between days 2 and 22 of exposure (Table 3). Concentrations of Ni in lungs of rats in the 2.5 mg

Table 3 Concentration of Ni in lungs of Ni&exposed Days NisS, exposure group (mg Ni3S2/m3) of expo- 0.6 2.5 sure fig Ni/lung pg Ni/g pg NiIlung control

DC 2.2 f 4.4 f 5.6 zt 6.3 zt 9.5 +

0.2 0.3 0.4 0.5 0.6

D 3.0 * 5.5 * 6.9 f 7.7 l 12 f

Ni3S2/m3 groups increased from 6.1 to 34 pg Nilg control lung between days 1 and 22 of exposure. Ni concentrations in lung did not increase linearly with time, rising rapidly during the first 7 exposure days, then more slowly, if at all, thereafter. Furthermore, lung concentrations (pg Ni/g control lung) were not always proportional to the Ni3S2 exposure concentration. 4.3. Biochemical and cytological changes in BALF Lactate dehydrogenase activity, an indicator of cytotoxicity, increased in the Ni3S2-exposed rats in an exposure-concentration-dependent manner (Fig. 2). LDH was significantly increased in rats after only 1 day of exposure to 2.5 mg Ni3S2/m3 and showed further sharp increases through day 4 of exposure, after which the activity appeared to plateau. LDH activity in BALF from rats exposed to 0.6 mg Ni3S2/m3 remained well below the activity in rats exposed to 2.5 mg Ni3S2/m3, especially during the first 4 days of exposure. Statistically significant increases in LDH activity in rats exposed to 0.6 mg Ni3S2/m3 occurred only after day 4 of exposure. Increases in BG activity were more pronounced among rats exposed to 2.5 than to 0.6 mg Ni3S2/m3 (Fig 3A). BG activity in rats exposed to

rat?

7000

0.2 0.4 0.2 0.7 0.6

4.6 f 0.6 8.5 f 0.7 11.9 f 0.9 22.7 zk 2.2 20.1 l 0.7 28.6 zt 2.3

1

T

!:I p-

p 5000

pg Ni/g control

3

6.1 + 11 f 15 f 28 f 24 f 34 l

,,.... /.“’

!!,..,,,,( “.

2.5

ml+ ,,,...... ..,

f,.....‘“”

....,,,

iI l

H

I

75

lung

lungb

1 2 4 7 12 22

103 (1995) 9-22

0.6 0.5 1.0 1.9 0.8 1.2

0 m+m3

El

‘Results represent the mean + S.E.M. of six values (three male, three female). bConcentrations of Ni in lung are expressed in terms of g of control lung in order to correct for increases in lung weight resulting from pathology. ‘Quantities of Ni in lungs were detectable but below the limit of quantitation of the method (1.87 pg Ni/lung).

0

( 0

I

5

I

10 Days of

I

15

I

20

I 25

Exposure

Fig. 2. Lactate dehydrogenase activity in BALF of rats exposed to 0,0.6 or 2.5 mg Ni3S2/m3as a function of time. Data points represent the mean f S.E.M. of ten values. *Indicate values significantly different from control (P 5 0.05).

J.M. Benson et al. / Toxicology 103 (1995) 9-22

1

so

T

15

A

0

5

10 DAYS

15 OF

20

25

EXPOSURE

Fig. 4. Alkaline phosphatase activity in BALF of control and Ni&exposed rats. *Indicate values significantly different from control.

with greater increases occurring among rats exposed to 2.5 mg Ni&/m3 (Fig. 3B). After day 7 of exposure, TP concentrations decreased slowly in rats exposed to 2.5 mg Ni3S2/m3 and increased slightly in rats exposed to 0.6 mg Ni3Sz/m3, so by

A

O;

’ 10

I

I

15

20

1 25

Day8 of Exposun O]

Fig. 3. (A) Beta-glucuronidase activity in BALF of rats exposed to 0, 0.6 or 2.5 mg Ni&/m3. (B) Total protein concentrations in BALF of the same rats. Data points represent the mean f S.E.M. of ten values. *Indicate values significantly different from control.

2.5 mg Ni&/mJ was signitkantly elevated by day 4 of exposure and increased sharply after day 7 of exposure. In contrast, BG activity in rats exposed to 0.6 mg Ni&/m3 was significantly elevated above control values only after days 7 and 12 of exposure, peaking after day 7 of exposure and returning to control values by the end of the study. Large increases ,in the concentration of TP in BALF occurred between days 1 and 7 of exposure in rats exposed to both concentrations of Ni&,

0

I

I

I

I

5

10

15

20

DAYS

100

1

01

0

1 25

OF EXPOSURE

Homogenate

I

I

I

I

5

10

15

20

DAYS

I 25

OF EXPOSURE

Fig 5. Concentration of glutathione in BALF (A) and in lung tissue homogenates (B) in control and Ni&exposed rats. *Indicate values significantly different from control.

J.M. Benson et al. / Toxicology 103 (1995) 9-22

16

day 22 of exposure, similar TP concentrations were present in BALF from both Ni& exposure groups. AP activity was decreased to approximately 25% or less of control values in rats exposed to either Ni& concentration after day 1 of exposure and for the most part, remained depressed throughout the remainder of the study (Fig. 4). The reason for the spike in activity (to control values) observed in rats after 4 days of exposure to 2.5 mg Ni&/m3 is unknown. Changes in GSH concentrations in either BALF or lung homogenates occurred only among rats exposed to 2.5 mg Ni3Sz/m3. Concentrations of GSH in BALF were significantly increased only in rats exposed to 2.5 mg Ni&/m3 for 4 days (Fig. 5A). GSH concentrations in lung homogenates were significantly decreased only after 7 and 22 days of exposure (Fig. 5B). Concentrations of CP in BALF were significantly increased in rats exposed to 0.6 mg Ni3S2/m3 after 4 days of exposure and to 2.5 mg Ni3Sz/m3 after 12 days of exposure (Fig. 6). The increases in CP were not exposureconcentration-dependent. The numbers of TNC recovered in BALF from rats exposed to 0.6 and 2.5 mg Ni3S2/m3 were significantly elevated by day 2 of exposure (Table 4). At that time, similar numbers of TNCs were

00 0

6

10 DAYS

OF

15

present in BALF from both Ni& exposure groups. Increases in BALF TNCs were not exposure-concentration dependent, with greater increases often occurring among rats exposed to 0.6 mg Ni3S,/m3. Increases in the numbers of neutrophils were, much greater among rats exposed to 2.5 mg Ni3Sz/m3 than 0.6 mg Ni3S2/m3. Significant increases in the numbers of neutrophils were observed after days 1 and 4 of exposure in rats exposed to 2.5 and 0.6 mg Ni,S,/m’, respectively. Some increases in numbers of AM in BALF were observed in the rats exposed to 0.6 mg Ni3S2/m3

Days of exposure 1

12

I

22

2

4

6.1 zt 0.4 9.2 zt 0.6b 10.3 f 0.9b

5.1 f 0.3 7.8 f 0.4b 10.6 f 0.6b

5.1 f 0.4 9.4 f O.gb 5.3 k 0.6

4.8 zt 0.5 12.1 l 0.7b 11.7 l 1.3b

4.5 zt 0.6 7.8 zt 0.9b 6.7 f 0.9

2.2 f 1.1 9.9 f 0.3 124 f 26b

1.3 l 0.7 25.6 + 0.3b 429 f 52b

2.5 l 0.9 244*44b 119 f 206

4.1 zt 1.6 173 f 22b 327 f 42b

1.0 l 0.7 116 zt 21b 220 l 47b

5.1 l 0.4 8.6 zt 0.6b 8.4 l O.lb

5.0 * 0.3 7.3 * 0.4b 5.5 f 0.7

4.6 f 0.4 6.5 zt 0.6b 4.0 + 0.4

4.6 + 0.5 9.9 zt 0.8b 8.0 l 1.2b

4.3 f 0.6 6.3 zt 0.8 4.1 f 0.5

Total nucleated cells (X 106)

0 0.6 2.5

7.0 f 0.5 6.4 zt 0.8 7.5 f 0.7

Total neutrophils (x 104) 0

1.5 f 1.1 3.4 f 1.4 11.2 zt 2.6b Total macrophages (X 106) 6.8 f 0.6 0 6.0 zt 0.8 0.6 6.9 f 0.6 2.5

0.6 2.5

25

Fig. 6. Concentration of collagenow peptides in BALF of rats exposed to Ni& for up to 22 days. *Indicate values significantly different from control.

Table 4 Summary of cytological changes in rats exposed to Ni$Sza N&S2 exposure group (ms/‘m3)

20

EXPOSURE

*Results represent the mean f S.E.M. of ten values. bMean significantly greater than control. Analysis of variance adjusted for multiple comparisons, P s 0.05.

1. M. Benson et al. / Toxic&gy 103 (1995) 9-22 Table 5 Effect of Ni& exposure on alveolar macrophage viability (percent viable)a Ni,S, exposure Percent viable group (mtim3) Days, of exposure 1 0

0.6 2.5

89 81 69

l

2 5 l 4b

l

2

4

7

12

22

89 + 2 74 l 8 46 f 6b

88 * 1 72 l 4b 56 f 2b

91 l 0.7 78 f 3b 69 zk 6b

80 * 4 93 * 1 81 +2

94 l 0.5 75 f 2b 65t lb

‘Results represent the mean f S.E.M. of five values, except in the case of rats exposed to 2.5 mg/m3 on days 7 and 22, where n = 2. bMean significantly below that of controls. Analysis of variance adjusted for multiple comparisons, P s 0.05.

for 2 to 12 da.ys. There was no exposure concentration-effect relationship, however, and numbers of AM present in BALF of rats exposed for 22 days were not different from control values. The viability of AM was strikingly reduced in rats exposed to 2.5 and 0.6 mg Ni&/m3 after days 1 and 4 of exposure, respectively (Table 5). Except at Day 12, macrophage viability in the Ni3S2exposed rats remained significantly below control values thereafter. 4.4. Morphological’ changes in rats exposed to 2.5 mg Ni3Sz/m3 Morphological changes occurring in the lung and nose are summarized in Table 6. One and 2 days after initiation of exposures, there was only a minimal increase in the numbers of AMs in the lung. These AMs were scattered throughout the lung and not aggregated near the terminal airways. Inflammatory lesions in the lung were most severe after day 4 of exposure. At this time, the alveolitis present was characterized by an increased number of AMs and neutrophils in the alveoli. The alveolar septa were slightly thickened with inflammatory cells. The inflammatory lesions were not restricted to the centriacinar region, but were relatively evenly distributed throughout the lung. In some foci, the intlammation resulted in alveolar deposition of protein and fibrin. Also after day 4 of exposure, moderate hyperplasia of bronchiolar epithelial &Is thickened the epithelium. Numerous infhunmatory cells were present in the

bronchial lumen, but seemed to have originated from the alveoli. After day 7 of exposure, alveolitis and bronchial epithelial hyperplasia were present, but not as severe as after day 4 of exposure. Proteinaceous debris in the alveoli was more prevalent after day 7 of exposure. This debris was associated with hypertrophic type II epithelial cells and with large vacuolated AM. These changes were designated alveolar proteinosis. After days 12 and 22 of exposure, the alveolitis was widely distributed through all alveoli. The severity of this lesion had not increased with time, however. The severity of alveolar proteinosis continued to increase with exposure duration and became more concentrated in the centriacinar region. Hypertrophic, vacuolated alveolar type II cells were present in the areas of proteinosis as were large degenerative macrophages. Bronchiolar epithelial hyperplasia was no longer present. After 22 days, a dark granular pigment, probably Ni&, was present in the cytoplasm of some macrophages. The patterns of changes in the severity of lung lesions with exposure duration in rats exposed to 2.5 mg Ni3$/m3 are depicted in Fig. 7. Nasal lesions were not striking. Slight, acute inflammation was present in the cuboidal and respiratory epithelium after day 2 of exposure. Minimal degeneration of the olfactory epithelium was noted after day 4 of exposure. This degeneration was characterized by a reduction of the volume of the apical cytoplasm in the epithelium caused by a

18

J.M.

Benson et al. /Toxicology

103 (1995) 9-22

Table 6 Incidencea and severity of lung and nasal lesions in rats inhaling Ni& mg Ni&/m3

Day 1 sacrifice Alveolar macrophage hyperplasia Day 2 sacrifice Alveolar macrophage hyperplasia Inflammation, nasal respiratory epithelium Day 4 sacrifice Bronchial epitheIia1 hyperplasia Alveolitis Alveolar proteinosis Degeneration, olfactory epithelium Day 7 sacrifice Bronchiolar epithelial hyperplasia Alveolitis Type 11 cell hypertrophy Alveolar proteinosis Atrophy, olfactory epithelium Degeneration, olfactory epithelium Day 12 sacritice Alveolitis Type II cell hypertrophy Alveolar proteinosis Degeneration, olfactory epithelium Inflammation, cuboidal epithelium Day 22 sacrifice Alveolar macrophage hyperplasia Alveolitis Type II cell hypertrophy Alveolar proteinosis Atrophy, olfactory epithelium Degeneration, olfactory epithelium Mucous cell hyperplasia Metaplasia, cuboidal epithelium

0

0.6

2.5

O/6

l/6 (1)

616 (1)

_b -

-

516 (1.2) 114(1)

-

-

6/6 (3.3) 6/6 (2.8) 416 (1.2) II6 (2)

-

116(1) 616 ( 1.4) l/6 (2) O/6 l/4 (I) II4 (1)

2l6 (1.8) 616 (1.8) 416 (2) 516 (1.8) l/4 (2) 314(1.7)

-

-

616 (2.2) 616 (1.3) 6/6 (2.0) 4/4 (1.8) l/4 (I)

l/6 (1) O/6 O/6 O/6 O/5 O/5 015 o/5

O/6 616 (1.8) 316 (1.5) 516(1.3) l/4 (I) 314(1.8) l/4 (1) o/4

O/6 616 (2.4) 616 (2.5) 616 (2.7) 214 (2.5) 214 (2.5) 314 (2) l/4 (2)

*Incidence is the number of rats having the lesion/number of rats examined. The mean severity score for those rats with the particular lesion is given in parenthesis. Data for male and female rats are combined. b , No tissues examined at this level at this sacriftce.

disorganization of the nuclear layer of the epitheliurn. Cytolysis was not prominent. The incidence of this subtle degenerative change was increased after day 7 and 12 of exposure. By day 22 of exposure, atrophy of the olfactory epithelium was only present in a few rats, resulting in an - 50% reduction in the thickness of the epithelium. 4.5. Morphological mg Ni$_Jm’

changes in rats exposed to 0.6

Morphological changes in lungs and nasal tissue

of rats exposed to 0.6 NijS2/m3 were qualitatively similar to those observed among rats exposed to 2.5 mg Ni3S2/m3, but were, in most cases less severe. 5. Discussion Nickel subsulfide, when repeatedly inhaled, is toxic in the respiratory tract of rodents. The toxic responses have been characterized morphologically and as biochemical and cytological changes

J.M. Benson et al. / Toxicology IO3 (199s) 9-22

i

i

li

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Days of Exposure

Fig. 7. Tends in severity of lung lesions in rats exposed to 2.5 mg Ni& for up to 22 days. Data points represent the mean of six values. Alveolar macrophage hyperplasia (a), bronchiolar epithelial hyperplasia (A), alveolitis (4 and alveolar proteinosis (+).

measured in BAL,F (Benson et al., 1989a, 1990; Dunnick et al., 1989). The purpose of this study was to determine the time-course of the incidence and severity of these types of changes occurring in F344/N rats inhaling 0.6 or 2.5 mg Ni&/m3, 6 h/day, 5 days/week: for up to 22 days. A secondary

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EXPOSURE

Fig. 8. Concentrations of Ni in lungs of rats exposed to 0.6 mg N&/m3 (circles) or to 2.5 mg N&l/m3 (triangles). Closed symbols are derived from Dunnick et al. (1989). Curves were fit to the data using the function f(x) = E(1 - emA?,where B is the predicted equilibrium concentration of Ni in lung, R is the Ni accumulation rate and t is time of exposure in days. Ni concentration in lung is expreclsedin terms of pg Ni/g control lung to correct for increases in lung weight due to pathology.

19

purpose was to quantitate Ni lung burdens as a function of time to obtain a better estimate of when lung concentrations reach equilibrium and, therefore, an estimate of the clearance half-time of Ni from the lungs of Ni3S1-exposed rats. Nickel concentrations in lung increased rapidly over the first 7 days of exposure and less rapidly, if at all, thereafter. Lung burdens in rats exposed to 0.6 or 2.5 mg Ni3S2/m3 for 22 days were 11.5 and 34.2 kg Ni/g control lung. These lung burdens are not statistically different (analysis of variance, P 6 0.05, adjusted for multiple comparisons) from those present in F344M rats exposed to Ni& aerosols under the same exposure conditions for 47 or 65 days (Dunnick et al., 1989) (Fig. 8), suggesting that equilibrium concentrations may be reached by 22 exposure days. Lung burdens resulting from exposure to 0.62 or 2.5 mg Ni&/m3 for l-65 days could be described by the function f(x) = B(1 - eMAt) where B = the equilibrium concentration of Ni in lung, A = the rate of Ni accumulation in peg/day and t = exposure time in days. Using this function, we predict that in rats exposed to 0.62 mg Ni&/m3, equilibrium concentrations of 12.8 pg Nifg control lung (95% confidence intarval = 9.6- 15.9 pg Ni/g control lung) would be achieved at an accumulation rate of 0.09 pg Ni/g control lung/day (0.02-o. 15 pg Ni/g control lung/day). For rats exposed to 2.5 rg Ni3Sz/m3, the predicted Ni equilibrium concentration in lung is 31 pg Ni/g control lung (25.8-36.6 pg Ni/g control lung), with an accumulation rate of 0.20 ag Ni/g control lung/day (0.07-0.33 pg Ni/g lung/day). The predicted equilibrium concentrations are close to achieved values (Fig. 8). Based on the Ni accumulation rates, we would predict half-times accumulation (and clearance) of Ni from Ni3Srexposed rat lungs of 3.5-8 days. This range of values is consistent with a pulmonary clearance half-time of -4 days reported for radioF344AU rats inhaling acutely labeled Ni& (Benson et al., 1994). By comparison, the half-time for clearance of soluble nickel sulfate hexahydrate from lung is l-3 days

20

J.M. Benson et al. I Toxicology 103 (1995) 9-22

(Medinsky et al., 1987). Inhaled Ni& then appears to have lung clearance properties characteristic of a soluble, rather than an insoluble, Ni compound. The changes observed in LDH, BG, TP and cytological parameters indicate that inhaled Ni3S2 produces cytotoxic and inflammatory changes in lung after only a few days of exposure. The cytotoxic effects, manifested in increases in LDH and decreased AM viability, appeared slightly earlier than the inflammatory changes (increases in BG, TP and neutrophils). The magnitude of the biochemical and cytological changes and the time to their onset were, for the most part, dependent on Ni& exposure concentration. The lack of direct correlation between the magnitude of the increases in BG activity in BALF and the numbers of neutrophils in BALF suggests that BG activity in BALF may be more a function of AM activation than of the numbers of macrophages present in alveoli recovered in BALF. This agrees with earlier data indicating that neutrophils are not the source of BG in BALF from rats exposed to cx quartz (Henderson et al., 1991). Inhalation of Ni3S2 inhibited AP activity immediately and in a nondose-dependent manner. AP activity in BALF can be decreased due to damage of type II epithelial cells or by direct inactivation of the enzyme by a toxicant. The cause of decreased AP activity in this study is not clear; however, morphological examination of lung indicated that the source of AP in lung, the type II epithelial cells, was damaged by Ni$z inhalation. Decreased activity also may be due to direct inhibition of the enzyme by Ni in BALF. While data are not currently available on the ability of Ni to inhibit lung AP activity, other metals have been shown to modulate lung AP activity in vitro and in vivo (Gupta et al., 1987; Boudreau et al., 1989). Low concentrations of Cd, Co and Mn inhibit lung AP in vitro, while high concentrations appear to activate the enzyme (Gupta et al., 1987). Inhaled CdC12 (5 mg/m3 for 1 h) has a multiphasic effect on lung AP activity recovered in BALF, with activity inhibited at 1 and 16 days postexposure and activated at 4 days post-exposure (Boudreau et al., 1989). On the other hand, rats inhaling Ga,O, (23 mg/m3, 2 h/day, 5 days/week,

for 4 weeks) had significantly elevated lung AP levels in BALF up to 12 months post-exposure (Wolff et al., 1988). Alveolar macrophages have been postulated as a source of GSH in acellular BALF (Boehme et al., 1992a,b). GSH may be actively released from macrophages during phagocytosis (Rouzer et al., 1982; Boehme and Henderson, 1990; Boehme et al., 1992b) or may be released from dead or dying macrophages and/or other cells in the alveolar region (Boehme and Henderson, 1990; Boehme et al., 1992). In support of this, increases of GSH in BALF from rats exposed to diesel exhaust for 18 months were significant, at the same time LDH activities in BALF peaked; however, GSH was increased to a lesser extent than LDH (Henderson et al., 1988). In this study, the pattern of increase in GSH in BALF did not parallel increases in LDH activity. GSH was significantly elevated (by approximately 2.5fold) only in BALF from rats exposed to 2.5 mg Ni3S2/m3 for 4 days (when histopathological lesions were most severe), while approximately 8- to lo-fold increases in LDH activity occurred in rats exposed to 2.5 mg Ni3S2/m3 for 4-22 days. As in the study of Henderson et al. (1988), the increase in GSH at 4 days of exposure was small compared to the corresponding increase in LDH activity. Our data do not support the idea that GSH is released from phagocytizing or dying macrophages, because increased levels of GSH in lavage fluid were not associated with the presence of large numbers of macrophages, often with significantly reduced viability. Significant decreases in tissue GSH after 7 and 22 days of exposure also suggest that, in addition to causing cellular damage in the lung, Ni may impair the ability of the lung to protect itself against oxidant damage through impaired synthesis of GSH or reduction of oxidized glutathione to GSH. CPs in BALF are soluble hydroxyprolinecontaining compounds that have leaked into the alveoli and small airways. Their presence in BALF suggests accelerated turnover of the extracellular collagenous matrix and probably correlates with some alteration of the pulmonary architecture. Increases may be related to an increase in enzymes capable of inducing collagenolysis such as polymorphonuclear leukocyte elastase and colla-

J.M. Betwonet al. /Toxicology IO3 (1995) 9-22

genase in polyrwrphonuclear leukocytes and AM. The increases in C:Pobserved in this study are consistent with results obtained in earlier studies in which rats exposed to 0.6 or 2.5 mg Ni&/m3 for 12 or 65 days had elevated CP in BALF (Benson et al., 1992). Increases in BALF CP have been associated with the development of pulmonary fibrosis in rats exposed to diesel exhaust (Henderson et al., 1988) but no fibrosis was detected in lungs of rats exposed for either 22 days (this study) or for 65 days (Dunnick et al., 1989; Benson et al., 1990). Chronic inhalation exposure of rats to Ni3S2 may be required for development of pulmonary fibrosis. Lung lesions associated with N&S, inhalation in this study, including AM hyperplasia, inflammation and alveolar proteinosis, were all present within the first 2-7 days of exposure. These changes were consistent with the large increases in BG activity, TP concentration and loss of AM viability determmed in BALF. However, interstitial infiltrates of mononuclear cells, observed in rats sacrificed after 65 days of Ni3S2 inhalation, were not observed among rats in this study, suggesting that the infiltration of mononuclear cells is a later response to Ni inhalation (Dunnick et al., 1989; Benson et al., 1990). Degeneration of the nasal olfactory epithelium was observed among rats on this study after day 4 of exposure and progressed to atrophy of the olfactory epithelium by day 22 of exposure. Atrophy of the olfactory epithelium was observed among rats exposed to 0.30-2.5 mg Ni&m3 for 65 days (Dunnick et al., 1989; Benson et all., 1990). In summary, inhaled Ni& produces cytotoxic and inflammatory effects in the lung characterized biochemically, cytologically and morphologically within only a few days of exposure. For the most part, the types of ‘changes observed in this study closely parallel those observed following 65-day exposures of rats tlo Ni3S2. When Ni lung burden data from this and the 65-day study are considered as a whole, it appears that Ni$$ is fairly soluble in lung, clearing with half-times in the range of - 3.5-8 days. Acknowledgements This research w,as conducted under Funds in

21

Agreement No. DE-FIO4-87AL44742 between the US. Department of Energy Office of Health and Environmental Research (Contract No. DEACO4-76EV01013) and the Nickel Producers’ Environmental Research Association, Inc. The facilities used for this research were fully accredited by the American Association for the Accreditation of Laboratory Animal Care. The authors gratefully acknowledge the technical contributions of J. Holmes, A. Magallanez, B. Tibbetts, J. Waide and R. White. References Benson,J.M., Burt, D.G., Cheng, Y.S., Hahn, F.F., Haley, P.J., Henderson, R.F., Hobbs, C.H. Pickrell, J.A. and Dunnick, J.K. (1989a) Biochemical responses of rat and mouse lung to inhaled nickel compounds. Toxicology, 57, 255-266. Benlon, J.M., Eidson, A.F., Hanson, R.L., Henderson, R.F. and Hobbs, C.H. (1989b) A rapid digestion method for analysis of nickel compounds in tissue by electrothermal atomic absorption spectrometry. J. Appl. Toxicol. 9, 219-222. Benson, J.M., Burt, D.G., Cheng, Y.S., Eidson, A.F., Gulati, K.K., Hahn, F.F., Hobbs, C.H. and Pickrell, J.A. (1990) Subchronic inhalation toxicity of nickel subsulfide to rats and mice. Inhal. Toxicol. 2, I-19. Benson, J.M., Burt, D.G., Cheng, Y.S.. Hahn, F.F., Haley, P.J., Henderson, R.F., Hobbs, C.H., Pickrell, J.A. and Dunnick, J.K. (1992) Effects of inhaled nickel on lung biochemistry. In: E. Nieboer and J.O. Nriagu (Eds), Nickel and Human Health, Wiley, New York, 1992, pp. 451-465. Benson, J.M., Barr, E.B., Bechtold, W.E. and Cheng, Y.S. (1994) Fate of inhaled nickel oxide and nickel subsulfide in F344/N rats. Inhal. Toxicol. 6, 167-183. Boehme D.S. and Henderson, R.F. (1990) Glutathione release by pulmonary alveolar macrophages in vitro: A possible index of particle cytotoxicity. Toxicologist 10, 277. Boehme, D.S., Maples, K.R. and Henderson, R.F. (1992a) Glutathione release by pulmonary alveolar macrophages in response to particles in vitro. Toxicol. Lett. 60, 53-60. Boshme, D.S., Hotchkiss, J.A. and Henderson, R.F. (1992b) Glutathione and GSH-dependent enzymes in bronchoalveolar lavage fluid cells in response to ozone. Exp. Mol. Pathol. 56, 37-48. Boudreau, J., Vincent, R., Nadeau, D., Trottier, B., Foumier, M., Krzystyniak, K. and Chevalier, G. (1989) The response of pulmonary surfactant-associated alkaline phosphatase following acute cadmium chloride inhalation. Am. Ind. Hyg. Assoc. J. 50, 331-335. Cohn V.H. and Lyle, J. (1966) A fluorometric assay for glutathione. Anal. B&hem. 14, 434-440. Dunnick, J.K., Elwell, MR., Benson, J.M., Hobbs, C.H., Hahn, F.F., Haley, P.J., Cheng, Y.S. and Eidson, A.F. (1989). Lung toxicity after 13-week inhalation exposure to

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J.M. Benson et al. / Toxicology 103 (1995) 9-22

nickel oxide, nickel subsulfide or nickel sulfate hexahydrate in F344/N rats and B6C3Ft mice. Fundam. Appl. Toxicol. 12, 584-594. Grant, R.A. (1965) Estimation of hydroxyproline by the autoanalyzer. J. Clin. Pathol. 17, 685-686. Gupta, S., Pandey, SD., Misra, V. and Viswanathan, P.N. (1987) Alkaline phosphatase as a model for studying metalprotein interactions in pulmonary toxicity. Environ. Res. 43, 24-30. Henderson, R.F., Rebar, A.H. and Denicola, D.B. (1979) Early damage indicators in the lungs. IV. Biochemical and cytological response of the lung to lavage with metal salts. Toxicol. Appl. Pharmacol. 51, 129-135. Henderson, R.F., Pickrell, J.A., Jones, R.K., Sun, J.D., Benson, J.M., Mauderly J.L. and McClellan, R.O. (1988) Response of rodents to inhaled dilute diesel exhaust: Biochemical and cytological changes in bronchoalveolar lavage fluid and in lung tissue. Fundam. Appl. Toxicol. II, 546-567. Henderson, R.F., Harkema, J.R., Hotchkiss, J.A. and Boehme, D.S. (1991) Effect of blood leukocyte depletion on the inflammatory response of the lung to quartz. Toxicol. Appl. Pharmacol. 109, 127-136. Keith, L.H., Crummett, W., Degon Jr, J., Libby, R.A., Taylor, J.K. and Wenther, G. (1983) Principles of environmental analysis. Anal. Chem. 55, 2210-2218. Mastromatteo, D. (1986) Nickel. J. Am. Ind. Hyg. Assoc. 47, 589-601.

Medinsky, M.A., Benson, J.M. and Hobbs, C.H. (1987) Lung clearance and disposition of 63Ni in F344/N rats after intratracheal instillation of nickel sulfate solutions. Environ. Res. 43, 168-178. Neuman, R.E. and Logan, M.A. (1950) The determination of hydroxyproline. J. Biol. Chem. 184, 299-305. Ottolenghi, A.D., Haseman, J.K., Payne, W.W., Falk, H.L. and MacFarland, H.N. (1974) Inhalation studies of nickel sulfide in pulmonary carcinogenesis of rats. J. Natl. Cancer Inst. 54, 1165-1172. Report of the International Committee on Nickel Carcinogenesis in Man. (1990) Stand. J. Work Environ. Health, 16, l-82. Rouzer, CA., Scott, W.A., Grifftth, O.W., Hamill, A.L. and Cohn, Z.A. (1982) Glutathione metabolism in resting and phagocytizing peritoneal macrophages. J. Biol. Chem. 257, 2002-2008. Sun, J.D., Pickrell, J.A., Harkema, J.R., McLaughlin, S.I., Hahn, F.F. and Henderson, R.F. (1988) Effects of buthionine sulfoximine on the development of ozoneinduced pulmonary fibrosis. Exp. Mol. Pathol. 49, 254-266. Wolff, R.K., Henderson, R.F., Eidson, A.F., Pickrell, J.A., Rothenberg, S.J. and Hahn, F.F. (1988) Toxicity of gallium oxide particles following a 4-week inhalation exposure. J. Appl. Toxicol. 8, 191-199.