(CANCER RESEARCH55, 5251-5256, November 15, 19951

that have shown an association between nickel exposure and cancer

(5, 6).We have previously reported the results of inhalation toxicity

studies to nickel compounds commonly found in the workplace,including nickel subsulfide, nickel oxide, or nickel sulfate. The nickeloxide used in these studies was “high-temperature―nickel oxide, atype of nickel that may be formed during high-temperature operationssuch as in the stainless steel industry. In the P344 rat and B6C3F1mouse, the major toxicity occurs in the lung after exposures to thesenickel compounds. Nickel sulfate caused lung toxicity at lower exposure concentrations than did nickel subsulfide or nickel oxide (7).Comparisons of the carcinogenic effects between nickel compoundsand differences in responses between rats and mice are described inthis paper, and information is provided for comparing results inexperimental models with those in humans.

MATERIALS AND METHODS

Chemicals. Nickel subsulfide (a Ni3S2;MW, 240.2; CAS No. 12035-72-2)and nickel oxide (NiO; green oxide, CalCined at 1350°C;MW, 74.7; CAS No.1313-99-1) were supplied by the International Nickel Co., Ltd. (Toronto,

Canada). Nickel sulfate hexahydrate (NiSO4@6H20;referred to as nickel salfate; MW, 262.9; CAS No. 10101-97-0)was obtained from Aldrich ChemicalCo. (Milwaukee, WI). Elemental analysis conducted on these compounds bythe Midwest Research Institute indicated overall purifies of 97—99%(8—10).The range of mass median aerodynamic diameters and o@obtained throughoutthe study of Ni3S2,NiO, and NiSO4-6H20 were 2.0—2.2@m(@52.0), 2.22.5p.m (o@1.8), and 2.2—2.5gun (o@2.2), respectively.

Aerosol Characteristics. The exposure systems and methods for aerosolgeneration and characterization have been described previously (8—10).Theanimals were exposed in Hazieton 1000 (mice) and Hazleton 2000 (rats)whole-body chambers (Lab Products, Inc., Maywood, NJ) for 6 h/day, 5

days/week, for 102 weeks. Ni3S2 and NiO aerosols were generated using 2'and 4' fluid bed generators, respectively. Nickel sulfate aerosols were generated by nebulization of nickel sulfate solutions. Aerosol concentration wasdetermined by taking three 2-h filter samples throughout the exposure day.Real-time determination of aerosol concentration was made using real timeaerosol monitor—modelS units. Aerosol size was determined using cascadeimpactors.

Experimental Design. Core groups of 50 male and 50 female F344 rats(Taconic Farms, Germantown, NY) and 50 male and 50 female B6C3F1mice(Simonsen Laboratories, Giroy, CA) were exposed to nickel sulfate hexahydrate, nickel subsulfide, or nickel oxide for 6 h/day, five days/week, for 2 years(Table 1).

The high exposure concentrations for nickel oxide and nickel sulfate wereselected based on the minimal to mild inflammatory lung lesions observed in

the 13-week toxicity studies (7). In the nickel subsuifide study, the highexposure concentration (1 mg/m3) corresponded to that used in a previous

nickel subsulfide study (11). The 0.15 mg/m3 concentration was selected based

on the severity of inflammatory lung lesions in the 13-week studies, whichcorresponded to that observed in the high exposure level selected for the 2-yearnickel oxide and nickel sulfate hexahydrate studies.

Additional animals were added for lung burden determinations at 7 or 15months. Atomic absorption spectroscopy was used for determining nickelburdens in the lung according to methods described previously (12).

Food (NIH-07 CertifIed Diet; Ziegler Brothers, Inc., Gardners, PA) wasprovided during nonexposure periods. Water was provided ad libitum. A 12-h

5251

Comparative Carcinogenic Effects of Nickel Subsulfide, Nickel Oxide, or NickelSulfate Hexahydrate Chronic Exposures in the Lung1

June K. Dunnick,2 Michael R. Elwell, Ann E. Radovsky, Janet M. Benson, Fletcher F. Hahn, Kristan J. Nikula,Edward B. Barr, and Charles H. Hobbs

National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 (J. K. D., M. R. E., A. E. R.j. and Lovelace inhalation Toxicology ResearchInstitute, Albuquerque. New Mexico 87185 Ii. M. B., F. F. H., K. J. N., E. B. B., C. H. H.]

ABSTRACT

The relative toxicity and carcinogenicity of nickel sulfate hexahydrate(NiSO4@6H2O),nickel subsulfide (Ni@S2),and nickel oxide (N1O) werestudied in F344/N rats and B6C3F1 mice after inhalation exposure for 6h/day, 5 days/week, for 2 years. Nickel subsultlde (0.15 and 1 mg/rn3) andnickel oxide (1.25 and 2.5 mg/rn3) caused an exposure-related Increasedincidence of alveolar/bronchiolar neoplasms and adrenal medulla neopla.stns in male and female rats. Nickel oxide caused an equivocal exposure-related increase in alveolar/bronchiolar neoplasms in female mice.

No exposure-related neoplastic responses occurred in rats or mice exposedto nickel sulfate or in mice exposed to nickel subsulfide. These findings areconsistent with results from other studies, which show that nickel subsultide and nickel oxide reach the nucleus in greater amounts than the dowater-soluble nickel compounds such as nickel sulfate. It has been proposed that the more water-insoluble particles are phagocytized, whereasthe vacuoles containing nickel migrate to the nuclear membrane, wherethey release nickel ions that effect DNA damage. The findings from theseexperimental studies show that chronic exposure to nickel can cause lungneoplasms in rats, and that this response is related to exposure to specific

types of nickel compounds.

INTRODUCTION

In 1932, the British Chief Inspector for Factories and Workshopsreported an increase in respiratory tract cancer in workers in the nickelrefinery in Wales (1). Further evidence that some hazard in the nickelrefinery caused these cancers was published some 25 years later, andexcess risks for respiratory cancer have been reported in other nickelindustries in Canada, Norway, and Russia (2, 3). Nickel is primarilyimported into the United States, where it is used in various industrialprocesses including fabrication of metal products, metal plating, andchemical and electric industries (4).

Cohorts of nickel-exposed workers continue to be studied to determine the parts and exposures of the refining processes that produce anincreased risk for cancer. However, it has not been possible to characterize specific risks from nickel exposures because exposures maybe multiple, including exposures to multiple nickel compounds, toother chemicals in the workplace, or to cigarette smoke or alcohol.Many of the studies that have found an association between nickelexposures and lung and/or nasal cancer have focused on “high-risk―cohorts. However, cancer was not found in all groups of exposednickel workers (5, 6). Excess mortality from nonmalignant respiratoryeffects or other disease have not been seen in epidemiological studies

Received 6/16/95; accepted 9/18195.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I This researchwas conductedunderInteragencyAgreementY0i-ES-30108 betweenthe National Institutes of Environmental Health Sciences and the United States Department of Energy. The in-life phase of the study was conducted at the Inhalation ToxicologyResearch Institute which is operated for the United States Department of Energy undercontract DE-ACO4-76EV01013. The facilities used for this research were fully accreditedby the American Association for the Accreditation of Laboratory Animal Care.

2 To whom requests for reprints should be addressed, at National Institute of Envi

ronmental Health Sciences, Research Triangle Park, NC 27709.3 The abbreviations used are: o@, approximate geometric SD; A/B, alveolar/bronchio

lar; it., intratracheal.

on March 29, 2019. © 1995 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

Table 1 Exposure level of nickel compound (or nickel; mg/rn3) in 2-year studies―

Nickel sulfate hexahydrate (22.3% nickel)Rats

CompoundNickel

MiceCompoundNickel

Nickel subsulfide (73.3% nickel)Rats

CompoundNickel

MiceCompoundNickel

Nickel oxide (76.6% nickel)Rats

CompoundNickel

MiceCompoundNickel

a Threshold limit values: 1 mg NiJm3; 0.1 mg NiJm3 (soluble nickels).

day/night cycle was maintained. Body weight and clinical signs were recordedevery 4 weeks.

Pathology. Complete necropsies were done on all animals. At necropsy, allorgans and tissues were examined for grossly visible lesions, and all major

tissues and lesions were preserved in 10% neutral-buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for microscopic examination.

Statistical Evaluation. The majority of neoplasms were considered to beincidental to the cause of death or not rapidly lethal and were evaluated by

logistic regression analysis (13, 14). Tests of significance included pairwisecomparisons of each exposed group with controls and a test for overall

exposure-related trends. Organ and body weight data were analyzed usingparametric multiple comparison procedures (15, 16), and lung burden datawere analyzed using nonparametric multiple comparison methods (17, 18).The reported values were considered significant at the P < 0.05 level.

RESULTS

Survival and Body and Lung Weights

Survival of rats and mice exposed to nickel sulfate, nickel subsulfide, and nickel oxide were in general similar to those of controls.Final mean body weights in both rats and mice were similar or 5—10%lower than controls (Tables 2 and 3). Lung weights in exposedanimals were greater than controls, and this was considered to berelated to inflammatory lung reactions that occurred in response tothese nickel exposures. The lung weights in the nickel oxide- and

Female ratsSurvivalFinal mean body wei@ht―Absolute lung weight

7-Month interim evaluation15-Month interim evaluation

a Percent relative to controls.

b Organ weight in grams.Cp@

dp@ o.os.

Table 2 Survival, body weight,and lung weigh: inratsExposure

(mg/m3)Nickelsulfatehexahydrate

(22.3% nickel)Nickel subsulfide (73.5%nickel)Nickel oxide(78.6%nickel)00.1250.25

0300.151.000.621.22.5Male

ratsSurvivalFinal mean body wei@ht―Absolute lung weight

7-Month interim evaluation15-Month interim evaluation16/54

1.672.1216/55

99

1.622.4818/55

21/55101 98

1.65 1.892.50 3.Otf13/53

21/5398

1.87 2.38'2.27 3.31@18/53

85

345C

6.84'14/541.722.2015/53

100

1.852.1515/53

95

2.46―3.30'12152

93

2.59c4.09'

22/5217/5328/5329/5426/5325/5328/5321/5326/5320/5326/5497979496789692901.251.221.221.45―1.31i.75@2.59c1.141.31―1.65'1.78'@1.371.571.49i.82c1.522.52―4.14'1.561.792.41'3.02C

CARCINOGENIC EFFECTS OF NICKEL EXPOSURE IN ThE LUNG

nickel subsulfide-exposed rats relative to controls were increasedmore than in mice and correlated with a more severe inflammatoryand toxic response in the lung of rats at the exposure concentrations

0 0.125 0.25 0.5 .0 0.03 0.06 0.1 1 used in these 2-year studies.

The increase in lung weights of animals in the nickel subsulfide and0 0.25 0.5 1.0 nickel oxide studies was greater than in the nickel sulfate studies and0 0.06 0.11 0.22 .

correlated with a more severe inflammatory response after exposuresto nickel subsulfide and nickel oxide. Nickel sulfate was more acutely

0 0.15 1 toxic than nickel subsulfide or nickel oxide, causing death of animals

0 0.11 0.73 at exposure concentrations of 2 mg/m3 in rats and 7 mg/m3 in mice as

0 0.6 1.2 has been reported previously in subchronic studies (7).

0 0.44 0.9 At 15-months, the lung weight in the high-exposure nickel sulfate

mice was 30—37%more than control lung weight, and in the rats,0 0 62 1 25 2 5 33—41% more than controls; the high-exposure lung weight in the

0@@ 2.0 nickel subsulfide mice was 78—92% more than controls, and in rats,

300% more; in the nickel oxide studies the high-exposure lung weight@ @5@ 5.0 in mice was 36—65% more than controls, and in rats, 86—94% more.

Noncarcinogenic Effects

A spectrum of exposure-related nonneoplastic respiratory tract lesionsseen after exposure to each of the nickels included focal alveolar/bronchiolar hyperplasia, inflammation, and/or fibrosis of the lung and lymph

oid hyperplasia of the lung-associated lymph nodes. Atrophy of theolfactory epithelium was also seen after exposure to nickel sulfate hexahydrate. Pigment (thought to represent nickel deposition) was also observed in the lung of animals exposed to nickel oxide. Qualitatively, theinflammatory responses of interstitial fibrosis and intraalveolar macrophage accumulation in the lung were similar in the three nickel compounds studied. However, at the exposure concentrations used, these

toxic lesions were considered to be more severe after exposure to nickeloxide and nickel subsullide. Mice were more resistant to development offocal hyperplastic and fibrotic lung lesions than were rats.

Carcinogenic Effects

Nickel Oxide Studies. There was an increase in the incidence ofA/B adenomas/carcinomas combined (A/B neoplasms) in male andfemale rats exposed to nickel oxide at concentrations of 1.2 and 2.5mg/m3 (Table 4). A slight increase in A/B adenomas occurred infemale mice exposed to 2.5 mg/m3. An increase in A/B adenomas/carcinomas combined occurred in female mice exposed to 1.2 mg/m3(Table 5).

The carcinogenic response in the lung of male and female ratsexposed to nickel oxide was related to exposure because (a) theincidence of A/B neoplasms was increased in both mid- and high

5252

on March 29, 2019. © 1995 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

Table 3 Survival, body weight.and lung weight inmiceExposure

(mg/m3)Nickelsulfatehexahydrate

(22.3% nickel)Nickel subsulfide (73.3%nickel)Nickel oxide(78.6%nickel)00.250.5

100.61.201.252.55Male

miceSurvivalFinal mean body wei@ht―Absolute lung weight

7-Month interim evaluation15-Month interim evaluation26/61

0.210.2423/61

94

0.200.2524/62

25/6197 91

0.22 0.230.26 O.31@26/61

25/5992

0.24 0.270.23 0.40'26/58

92

0.34c0.41'19/57

0.190.2323/67

93

0.210.2529/66

93

0.24c0.31'23/69

93

0.24'0.38'

34/6139/6045/6037/6038/5834/5938/6041/6440/6642/6338/6491948890869694900.220.210.220.250.19026d0.29c0.180.210.230.230.240.240.280.33'0.260.39'0.50'0.250.260.290.34c

Table 4Alveolar/bronchiolarneoplasms inratsNickel

sulfate hexahydrate(22.3% nickel)Nickel subsulfide (73.5%nickel)Nickel oxide(78.6%nickel)00.1250.250.50

0.151.000.621.252.554―

02'2―53

00053

01153

21353

530 30 30 6b53

6b7b

11d54

o53 I

i53

33oe52

22

4e52―

00053

00053

00054

10153

532 50 ice2 6@e53

549b53

1053

0053

15b6e54

4

@e

CARCINOGENIC EFFECTS OF NICKEL EXPOSURE IN THE LUNG

Female miceSurvivalFinal mean body wci@ht―Absolute lung weight

7-Month interim evaluation15-Month interim evaluation

a Percent relative to controls.

I, Organ weight in grams.

C p o.oi.

dp < o.os.

exposure groups relative to concurrent and historical control rates, and(b) the effects were observed in both male and female rats. A marginalincrease in A/B neoplasms was seen at the low and mid exposurelevels in female mice.

Nickel Subsulfide Studies. There was an increase in the incidenceof A/B adenomas, A/B carcinomas, and A/B adenoma/carcinomascombined in male rats exposed to 1 mg/m3. The incidences of A/Badenomas/carcinomas combined in male rats exposed to 0.15 mg/m3and in female rats exposed to 0.15 and 1 mg/m3 (Table 3) were alsoincreased. No exposure-related neoplasms were observed in miceexposed to nickel subsulfide (Table 5).

The carcinogenic response in the lung of male and female ratsexposed to nickel subsulfide was clear evidence for a carcinogeniceffect because neoplastic lung lesions were observed at both exposurelevels, the effect was observed in males and females, and the mcidence for these neoplasms was increased relative to concurrent andhistorical control rates.

Nickel Sulfate Studies. There were no increases in lung neoplasmsin rats or mice exposed to nickel sulfate (Tables 4 and 5). The A/Bneoplasms that were observed in the exposed groups were similar inincidence and morphology to those observed in controls.

Description of Lung Neoplasms

The A/B neoplasms in mice and in some rats in the nickel subsulfide and nickel oxide studies had morphology consistent with spontaneously occurring lung neoplasms in rodents. Typically, most A/Bcarcinomas had cuboidal or low columnar epithelium in mixtures oftubular, papillary, and alveolar growth patterns. Neoplastic cells incarcinomas were pleomorphic and often stained deeply basophilic.

Focal alveolar epithelial hyperplasia was part of the morphologicalcontinuum toward neoplasia and consisted of discrete areas wherealveoli were lined by cuboidal cells that occasionally formed smallprojections within alveolar lumens.

Some of the A/B neoplasms in rats exposed to nickel subsulfide ornickel oxide had morphological features that differed from the typicalpapillary or solid neoplastic proliferations of A/B epithelium typicallyfound in control rats. Different morphological features included aprominent fibrous tissue component (Fig. 1) and/or areas of differentiation to stratified squamous epithelium in A/B carcinomas (Fig. 2).In addition, inflammatory cells, cellular debris, and areas of squamousdifferentiation or a prominent dense fibrous tissue component occurred in some A/B adenomas.

Other Carcinogenic Effects

A carcinogenic response was also seen in the adrenal medulla ofnickel subsulfide and nickel oxide rats (Table 6). There were no othersites for exposure-related increases in neoplasms in rats or in miceafter exposure to nickel sulfate, nickel oxide, or nickel subsulfide.

Lung Burden Data

Analysis of the nickel lung burden data showed accumulation ofnickel in the lungs of rats and mice after exposure to nickel oxide,whereas with nickel subsulfide and nickel sulfate, nickel was clearedfrom the lungs. The lung burden at 7 or 15 months in rats and micewas 1—2@ Ni/g lung for nickel sulfate, 3—26p.g Ni/g lung for nickelsubsulfide, and 175—2,258p@gNi/g lung for nickel oxide (Table 7).

The lungs were enlarged particularly in the exposed groups of rats in

Exposure (mg/m3)

Male ratsAdenomaCarcinomaAdenoma/carcinoma

Female ratsAdenomaCarcinomaAdenoma/carcinoma

a Number of animals examined.

bp@ 0.05.( includes data for one squamous cell carcinoma.

dp < o.oi.e p < o.os vs. historical data [1.4%, 3/210 (males); 1.4%, 4/208 (females)].

5253

on March 29, 2019. © 1995 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

CARCINOGENIC EFFECTS OF NICKEL EXPOSURE IN ThE LUNG

p ..‘@;,-,@@ _% —@@,‘-‘-) ,- @‘@ . ‘ @ri@ ? .

@@ .;@v@ _@ v4@;@@ e@ a,@

p ‘@‘,“@I―@@1@@ ,@@ ‘;@@.

@ .-*,i@

r@ -@@@ “i.,-@':@@@@@ .,;@cw@?@ -@@@t,z:@4@,.. A'. 4 1 ,p@ @‘@@ ‘. ç@ -@ •@ . “I ,,@@ •@

- @4@/ ;?*7 $@)i@

@54@@ (@ ,

@)@4@.'p%@ ‘ :@.4@

Fig. 1. Alveolar/bronchiolar carcinoma from a male rat exposed to 0. 15 mg/m3 nickelsubsulfide for 2 years. Note the extensive connective tissue between the neoplasticepithelial cells. X240.

the nickel oxide and nickel subsulfide studies. Therefore, when the ratlung burden data at 7 or 15 months are expressed as amount of nickel pertotal lung, the nickel accumulation after nickel oxide exposures is emphasized: 1—2 @gNi/lung for nickel sulfate; 9—29 @gNi/lung for nickelsubsufide; and 226—4573 @gNi/lung for nickel oxide.

DISCUSSION

The most significant findings from these studies were that nickelsubsulfide and nickel oxide caused lung neoplasms in rats, and nickeloxide caused a marginal increase in lung neoplasms in mice (Table 8).There was also inflammation, hyperplasia, and fibrosis in the lungs ofrats, and to a lesser extent in mice, exposed to all three nickelcompounds. No exposure-related lung neoplasms occurred in rats ormice in the nickel sulfate studies.

In the nickel subsulfide rats, there were increases in lung neoplasmsat 0.15 and 1.0 mg/rn3 (0.1 1 and 0.73 mg Ni/m3) and in the nickeloxide rats at 1.25 and 2.5 mg/m3 (1.0 and 2.0 mg Ni/m3) after nickelexposures of more than 1 year. As a point of reference, the thresholdlimit value for insoluble nickel compounds is 1 mg Ni/rn3, althoughmost work situations would involve lower nickel exposures (19).

The type of nickel compound is important in the eventual carcinogenic response. Under the conditions of these studies, the nickelcompound that was more rapidly cleared from the lungs (nickelsubsulfide) gave a stronger carcinogenic response than the nickelcompound retained in the lungs (nickel oxide). Thus, the amount ofnickel in the lung (which represents the difference between theamount of nickel deposited in the lung and the amount removed by theclearance mechanisms) does not predict the severity of the lung tumorresponse. In these studies, nickel sulfate and nickel subsuffide werecleared from the lung, whereas nickel oxide accumulated. This agreeswith previous studies, which showed that the half-life of nickel sulfatein the rat lung is 1—3days (20), that of nickel subsuffide, 5 days, andthat of nickel oxide, approximately 120 days (21).

In humans, it has also been shown that the amount of nickelretained in the lung may not predict the carcinogenic response afternickel exposure. In lung tissue specimens from 39 workers in thenickel industry (22), the average nickel concentration for workers inroasting and smelting operations was 330 ±380 p.g nickel/g dry lungweight; for workers in electrolysis departments, 34 ±48 @@g;and forunexposed people, 0.76 ±0.39@ (dry lung represents approximately 20% “wet―lung weight, the lung weight used in our studies in

rodents). Workers who were diagnosed with lung cancer (14 cases)had the same lung nickel burdens at autopsy as did nickel workers (25cases) who died of other causes. This study also found that lungcancer occurred in workers from the electrolysis department (8 of 24),as well as those from the roasting and smelting operations (6 of 15),although those from the electrolysis department had lower lung nickelburdens. Thus, both our studies and the studies in humans show thata measurement of retained nickel does not necessarily predict theextent of lung cancer after nickel exposures.

The formation of lung neoplasms in rodents after exposures tonickel subsulfide and nickel oxide were considered to be specificallyrelated to the nickel exposures. Nickel subsulfide lung burdens at 15months remained below 30 @gNi/g lung, but nickel lung burdensincreased in the nickel oxide studies to approximately 1000 @gNi/glung (>4000 @gNi/lung). The possible relationship between nickelaccumulation in the lung in the nickel oxide studies and the toxicological effects remains unclear at this time. Recent studies have shownthat repeated exposure to 0.62 and 2.5 mg NiO/m3 results in impairedclearance of subsequent inhaled nickel oxide (23).

Rat carcinogenicity studies with other relatively nontoxic particles,such as titanium dioxide [250 mg/m3 (24)J, talc [18 mg/m3 (25)], andantimony trioxide [45 mg/m3 (26)], required higher aerosol concentrations to produce a carcinogenic response than did the nickel compounds. For example, the carcinogenic response in the rat lung in talcstudies was seen at 18 mg/m3, and at this exposure, lung burden wasapproximately 25 mg talc/g lung at 18 and 24 months. The lung tumorresponse with these other substances may be due in part to accumulation of nontoxic particles.

Other studies have shown that nickel subsuffide is carcinogenic in therat after local injection (5), inhalation exposure (1 mg/m3) (11), and i.t.administration (27, 28), but not after i.t. administration in hamsters (29).

Nickel oxide has been shown to be carcinogenic after local injection inrodents, but there have been no previous chronic inhalation studies withthis compound in rats. One long-term nickel oxide inhalation studyperformed in hamsters (50 mg/m3) was negative for carcinogenic activity(30). In contrast,nickelsulfatedoesnot producetumorsafterlocalinjection (5). Therefore, it appears that for the nickel compounds used inthese studies, the carcinogenic response after local injection predicts thecarcinogenic response after inhalation exposure in the rat.

Selection of the model system is important in assessing and predicting the potential extent of environment disease. More chemicalrelated lung neoplasms were observed in nickel subsulfide- and nickeloxide-exposed rats than in mice. In studies of other metals (e.g.,

.@ @@y?,@'H:.

•@M@@4g@.

@@ .

‘@ @.

\. ‘.4.@ @-:

@,@ .. :4-

... ;- ‘

...

*i@..i@ @..

...“o V-.-.

Fig. 2. Alveolar/bronchiolar carcinoma with areas of squamous differentiation from amale rat exposed to 1.0 mg/m3 nickel subsulfide. X 150.

5254

on March 29, 2019. © 1995 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

Table 5 Alveolar/bronchiolar neoplasms inmiceNickel

sulfate hexahydrate (22.3% nickel) Nickel subsulfide (73.3% nickel) Nickel oxide (78.6%nickel)Exposure

(mg/in3) 0 0.25 0.5 1 0 6 1.2 0 1.25 2.55Male

mice 61― 61 62 61 61 59 58 57 67 6669AdenomaS 5 3 5 6 3 2 7 5 611Carcinoma9 13 4 3 7 2 4 4 10 96Adenoma/carcinoma

13 18 7 8 13 5 6 9 14 1514Female

rats 61― f,4J f@ f@f@ 58 59 60 64 66 63 64Adenoma 3 3 2 0 3 1 1 2 4 lO@3Carcinoma

4 3 9 2 7 1 2 4 11 42Adenoma/carcinoma7 6 10 1 9 2 3 6 l5'@ 128a

Number of animalsexamined.bp< o.os.Table

6 Adrenal medulla prol(ferative lesions inratsNickel

sulfate hexahydrate (22.3% nickel) Nickel subsulfide (73.3% nickel) Nickel oxide (78.6%nickel)Exposure

(mg/m3) 0 0.125 0.25 0.5 0 0.15 1.0 0 0.62 1.22.5Male

rats 54― 55 55 55 53 53 53 54 53 5354Hyperplasia28 20 18 26 26 22 10 25 27 26 24

Benign pheochromocytoma 16 16 12 11 13 30― 38b 27 24 26 32Malignant pheochromocytoma 0 3 2 1 0 2 10― 0 0 1 6cBenign or malignant pheochromocytoma 16 19 13 12 14 30― 42― 27 24 2735bFemale

rats 52a 53 53 54 53 53 53 53 53 5354Hyperplasia6 4 8 8 5 11 16b 8 12 14 22―

Benign pheochromocytoma 2 4 2 3 2 7 36b@@ 618―Malignantpheochromocytoma 0 0 0 0 1 0 1 0 0 0 0

Benign or malignant pheochromocytoma 2 4 2 3 3 7 36b@@ 618―a

Number of animalsexamined.bp

<o.oi.‘.

P@ 0.05.cadmium

compounds) exposure-related lung neoplasms have also Nasopharyngeal carcinoma in humans has been attributed tonickelbeenfound to occur more frequently in rats than in mice (31, 32). exposure (6). In our studies, there was no evidence of carcinogenesisinThus,these findings suggest that the rat may be more sensitive as a the nasal cavity in either rats or mice. The preponderance ofthesemodel

than the mouse for metal-induced toxic and carcinogenic sinonasai neoplasms in humans have been classified as anaplastic, undifresponses in the lung, and also a better model system for predicting ferentiated, or squamous cell carcinoma (33). In nickel sulfate (jredisease in humans. It should be noted that the mouse is more suscep- chronice and chronic) and nickel subsulfide (prechronic) inhalation rotible to the formation of other chemically-induced lung neoplasms dent studies (7), the olfactory epithelium, rather than the respiratory orfrom epoxide and halogenated chemicals (8—10). squamous mucosa, was the target site for chemical-related toxicity.

Whereas the relationship of increased adrenal medullary tumors Nickel sulfate was not carcinogenic in the lung of rats ormice.andexposure to nickel subsulfide or nickel oxide is clear, the mech- However, in prechonic studies at higher exposure, this compound

anism for this increase is uncertain. It is possible that a small amount resulted in mortality and necrotizing pneumonia at concentrations thatof nickel may reach the adrenal medulla and cause a direct carcino

were nonlethal with exposure to nickel subsulfide and nickel oxide. Itgenic effect at that site. There is some distribution of nickel subsulfide

has been found that exposure to the water-soluble nickel salts (i.e.,to extrarespiratory tissues (e.g. , kidney), but a significant increase in

nickel sulfate) may have a synergistic role with oxidic or other formsnickel was not detected beyond the respiratory tissues with nickelof nickel in causing lung and nasal neoplasms in nickel refineryoxide exposures. A similar increase in adrenal tumors has also been

seen in inhalation studies with an unrelated inert particle (talc). It is workers (6). Experimental studies have shown that water-solublepossible that an unknown secondary mechanism related to chronic nickel salts enhance the cytotoxicity and mutagenicity of DNApulmonary inflammation may be related to the observed increase in damaging agents by inhibiting DNA repair processes (34). The inhitumors in the adrenal medulla. bition of DNA repair may be one mechanism by which the water

Table 7 Lung burden analyses in the studies ofnickel sulfate hexahydrate, nickel subsulfide, and nickeloxideNickel

sulfate hexahydrate (22.3% nickel) Nickel subsulfide (73.3% nickel) Nickel oxide (78.6% nickel)

0 0.12 0.25 0.5 1 0 0.15 0.6 1 1.2 0 0.62 1.25 2.5 5

1b69—175388701———1—69—173477713—112—1011—1624421,034—122—1014—169533861———1—43—3287461,116———2—47—262706949—I12—1526—3319591,798—122—1220—4511,2372,258

CARCINOGENIC EFFECFS OF NICKEL EXPOSURE IN THE LUNG

Exposure (mg/m3)

7-Month interim evaluationMale ratsFemale ratsMale miceFemale mice

15-Month interim evaluationMale ratsFemale ratsMale miceFemale mice

a Results were below the limit of detection.

b Values represent mean amount of nickel (@xg nickel/g lung). Lung burden groups included 5—7 animals.

5255

on March 29, 2019. © 1995 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

Table 8 LeveIs of carcinogenicactivityNickel

sulfatehexahydrateNickelsubsulfideNickeloxideMale

ratsNoClear (lung, adrenalmedulla)Some

(lung, adrenalmedulla)Female

ratsNoClear (lung, adrenalmedulla)Some

(lung, adrenalmedulla)MalemiceNoNoNoFemale

miceNoNoEquivocal (lung)

CARCINOGENIC EFFECrS OF NICKEL EXPOSURE IN THE LUNG

14. Dinse, G. E., and Haseman, J. K. Logistic regression analysis of incidental tumor datafrom animal carcinogenicity experiments. Fundam. Appi. Toxicol., 6: 44—52,1986.

15. Dunnett, C. W. A multiple comparison procedure for comparing several treatmentswith a control. J. Am. Stat. Assoc., 50: 1096—1121, 1955.

16. Williams, D. A. The comparison of several dose levels with a zero dose control.Biometrics, 28: 519—531,1971.

17. Shirley, E. A non-parametric equivalent of Williams' test for contrasting increasingdose levels of a treatment. Biometrics, 33: 386—389, 1977.

18. Dunn, 0. J. Multiple comparisons using rank sums. Technometrics, 6: 241-251, 1964.19. American Conference of Govemmental Industrial Hygienists. Threshold limit values

for chemical substances and physical agents and biological exposure indices for1993—1994.Cincinnati, OH: American Conference of Governmental Industrial Hygienists, 1993.

20. Medinsky, M. A., Benson, J. M., and Hobbs, C. H. Lung clearance and disposition of“3Niin F3441Nrats after intratracheal instillation of nickel sulfate solutions. Environ.Res., 43: 168—178,1987.

21. Benson, J. M., Barr, E. B, Bechtold, W. E., Cheng, Y. S., Dunnick, J. K., Eastin,W. E., Hobbs, C. H., Kennedy, C. H., and Mapples, K. R. Fate of inhaled nickel oxideand nickel subsulfide in F344/N rats. Inhal. Toxicol., 6: 167—183,1994.

22. Andersen, I., and Svenes, K. B. Determination of nickel in lung specimens of thirty-nineautopsiednickel workers@lot. Arch. Occup. Environ. Health, 61: 289-295, 1989.

23. Benson, J. M., Chang, I., Cheng, Y. S., Hahn, F. F., Kennedy, C. H., Barr, E. B.,Maples, K. R., and Snipes, M. B. Particle clearance and histopathology in lungs ofF344/N rats and B6C3F1 mice inhaling fickle oxide or nickel sulfate. Fundam. Appl.Toxicol., in press, 1995.

24. Lee, K. P., Trolchimowicz, H. J., and Reinhardt, C. R. Pulmonary response of ratsexposed to titanium dioxide (Ti02) by inhalation for two years. Toxicol. Appl.Pharmacol., 79: 179—192,1985.

25. National Toxicology Program. Toxicology and carcinogenesis studies of talc inF344/N rats and B6C3F, mice. TR 421. NIH Publ. No. 93-3152. Research TrianglePark, NC: NIH, 1993.

26. Groth, D. H., Stettler, L E., Burg, J. R., Busey, W. M., Grant, G. C., and Wong, LCarcinogenicity effects of antimony trioxide and antimony ore concentrate in rats. J.Toxicol. Environ. Health, 18: 607—626, 1986.

27. PoU, F., Ziem, U., Reiffer, F. J., Huth, F., Ernst, H., and Mohr, U. Carcinogenicitystudies on fibres, metal compounds, and some other dusts in rats. Exp. Pathol., 32:129—152,1987.

28. Yarita, T., and Nettesheim, P. Carcinogenicity of nickel subsulfide for respiratorytract mucosa. Cancer Rca., 38: 3140—3145.

29. Muhle, H., Belimann, B., Takenaka, S., Fuhst, R., Mohr, U., and Pott, F. Chroniceffects of intratracheally instilled nickel-containing particles in hamsters. In: E.Nieboer and J. 0. Nriagu (eds.), Nickel and Human Health: Current Perspectives,pp. 467—479.New York: John Wiley and Sons, Inc., 1992.

30. Wehner, A. P., Busch, R. H., Olson, R. J., and Craig, D. K. Chronic inhalation of nickeloxide and cigarette smoke by hamsters. Am. Ind. Hyg. Assoc. J., 36: 801—810,1975.

31. Glaser, U., Hochrainer, D., Otto, F. J., and Oldiges, H. Carcinogenicity and toxicity offour cadmiumcompoundsinhaledby rats ToxicoLEnviron.Chem.,27: 153-162, 1990.

32. Heinrich, U., Peters, L, Ernst, H., Rigginghausen, S., Dasenbrock, C., and Konig, H.Investigation on the carcinogenic effects of various cadmium compounds after inhalation exposure in hamsters and mice. Exp. Pathol., 37: 253—258,1989.

33. Sunderman, F. W., Morgan, L. G., Andersen, A, Ashely, D., and Forouhar, F.Histopathology of sinonasal and lung cancers in nickel refinery workers. Ann. Clin.Lab. Sci., 19: 44—50,1989.

34. Hartwig, A., Mullenders, L. H. R., Schepegrell, R., Kasten, U., and Beyersmann, D.Nickel(ll) interferes with the incision step in nucleotide excision repair in mammaliancells. Cancer Rca., 54: 4045-4051, 1994.

35. Costa, M., Zhuang, Z., Huang, S., Cosentino, S., Klein, C. B., and Salnikow, K.Molecular mechanisms of nickel carcinogenesis. Sci. Total Environ., 148: 194—199,1994.

36. Costa, M., and Molienhauer, H. H. Carcinogenic activity of particulate metal cornpounds is proportional to their cellular uptake. Science (Washington DC), 209:515—517,1980.

37. Abbracchio, M. P., Heck, J. D., and Costa, M. The phagocytosis and transformingactivity of crystalline metal sulfide particles are related to their negative surfacecharge. Carcinogenesis (Land.), 3: 175—180,1982.

38. Abbracchio, M. P., Simmons-Hansen, J. S., and Costa, M. Cytoplasmic dissolution ofphagocytized crystalline metal sulfide particles: a prerequisite for nuclear uptake ofnickel. J. Toxicol. Environ. Health, 9: 663—676, 1982.

39. Evans, R. M., Davies, P. J. A., and Costa, M. Video time-lapse microscopy study ofphagocytosis and intracellular fate of crystalline nickel sulfide particles in culturedmammalian cells. Cancer Res., 42: 2729—2735,1982.

40. Costa, M. Molecular mechanisms of nickel carcinogenesis. Annu. Rev. Pharmacol.Toxicol., 31: 321—337,1991.

41. Huang, X., Klein, C. B., and Costa, M. Crystalline Ni3S2 specifically enhances theformation of oxidants in the nuclei of CHO cells as detected by dichlorofluorescein.Carcinogenesis (Land.), 15: 545—548,1994.

42. Lee, Y. W., Pons, C., Tummolo, D. M., Klein, C. R., Rossman, T. G., and Christie,N. T. Mutagenicity of soluble and insoluble nickel compounds at the gpt locus in G12Chinese hamster cells. Environ. Moi. Mutagen., 21: 365—371,1993.

43. Klein, C. B., Conway, K, Wang, X. W., Bhamra, R., Un, X., Cohen, M. D., Annab, L,Barrett@J. C., and Costa, M. Senescence of nickel-transformed cells by an X chromosome:possibleepigeneticcontrol. Science (WashingtonDC), 251: 796—799,1991.

44. Lee, Y. W., Klein, C. B., Kargacin, B., Sainikow, K., Kitahara, J., Dowjat, K.,Zhitkovich, A., Christie, N. T., and Costa, M. Carcinogenic nickel silences geneexpression by chrornatin condensation and DNA methylation: a new model forepigenetic carcinogens. MoI. Cell. Biol., 15: 2547—2557, 1995.

soluble nickel salts have a synergistic role in enhancingcarcinogenesis after exposure to other nickel compounds.

The mechanisms for how nickel compounds cause carcinogenic effectsis an area of continuing investigation. In vitro studies have shown thatwater-insoluble nickel compounds are more readily phagocytized thanthe water-soluble nickel compounds and, therefore, will reach the nucleusof the cell in greater amounts than will the water-soluble nickel compounds (35—40).Nickel subsulfide produces a high level of oxidants inthe nuclei of cells, which could directly damage protein and DNA (41,42). Other in vitro studies suggest that nickel may cause gene alterationsby enhanced DNA methylation and heterochromatin condensation, cansing inactivation of Critical tumor suppressor or senescence genes (43, 44).

Our findings of the carcinogenic response in the rat lung afterexposure to nickel subsulfide or nickel oxide agree with epidemiologyfindings, which show that exposure of workers particularly to theoxidic and sulfidic nickel compounds may lead to an increased risk oflung cancer (6). The epidemiology studies also suggest that exposureto soluble nickel salts enhances the risk associated with exposure to

5256

less soluble forms of nickel.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the contributions of the technical staffof the Inhalation Toxicology Research Institute and Des. Robert Goyer andRonald Herbert (National Institutes of Environmental Health Sciences) fortheir helpful discussions and review of the manuscript.

REFERENCES

1. Bridge, J. D. Annual Report of the Chieflnspector of Factories and Workshops for theYear 1932, pp. 103—104.London, UK: His Majesty's Stationery Office, 1933.

2. Doll, R. Cancer of the lung and nose in nickel workers. Br. J. md. Med., 15: 217—223,1958.

3. Morgan, J. 0. Some observations on the incidence of respiratory cancer in nickelworkers. Br. J. md. Med., 15: 224—234, 1958.

4. United States Bureau of Mines. Nickel. In: Mineral Commodity Summaries 1991, pp.110—111. Washington, DC: United States Department of the Interior, Bureau ofMines, 1991.

5. LARC. IARC Monographs on the Evaluation of Carcinogenic Risk to Humans.Chromium, Nickel and welding, Vol 49. Lyon, France: IARC, 1990.

6. Doll, R., Andersen, A., Cooper, W. C., Cosmatos, I., Cragle, D. L, Easton, D.,Enterline, P., Goldberg, M., Metcalfe, L., Norseth, T., Peto, J., Rigaut, J., Roberts, R.,Seilkop, S. K., Shannon, H., Speizer, F., Sunderman, F. W., Jr., Thornhill, P., Warner,J. S., Weglo, J., and Wright, M. Report of the international committee on nickelcarcinogenesis in man. Scand. J. Work Environ. & Health, 16: 1—82,1990.

7. Dunnick, J. K., Elwell, M. R., Benson, J. M., Hobbs, C. H., Hahn, F. F., Haley, P. J.,Cheng, Y. S., and Eidson, A. F. Lung toxicity after 13-week inhalation exposure tonickel oxide, nickel subsulfide, or nickel sulfate hexahydrate in F344/N rats andB6C3F, mice. Fundam. AppI. Toxicol., 12: 584—594, 1989.

8. National Toxicology Program. Toxicology and Carcinogenesis Studies of NickelSubsulfide. NIH PubI. No. 94-3369. Research Triangle Park, NC: NIH, 1994.

9. National Toxicology Program. Toxicology and Carcinogenesis Studies of NickelOxide. NIH PubI. No. 94-3363. Research Triangle Park, NC: NIH, 1994.

10. National Toxicology Program. Toxicology and Carcinogenesis Studies of Nickel SulfateHexahydrate. NIH PubI. No. 94-3370. Research Triangle Park, NC: NIH, 1994.

11. Ottolenghi, A. D., Haseman, J. K., Payne, W. W., Falk, H. L., and MacFarland, H. N.Inhalation studies of nickel sulfide in pulmonary carcinogenesis of rats. J. Natl.Cancer Inst., 54: 1165—1170,1975.

12. Benson, J. M., Eidson, A. F., Hanson, R. L, Henderson, R. F., and Hobbs, C. H. Arapid digestion method for analysis of nickel compounds in tissue by electrothermalatomic absorption spectrometry. J. Appl. Toxicol., 9: 219—222, 1989.

13. Dinse, 0. E., and Lagakos, S. W. Regression analysis of tumour prevalence data.Appl. Statist., 32: 236—248,1983.

on March 29, 2019. © 1995 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

1995;55:5251-5256. Cancer Res   June K. Dunnick, Michael R. Elwell, Ann E. Radovsky, et al.   LungOxide, or Nickel Sulfate Hexahydrate Chronic Exposures in the Comparative Carcinogenic Effects of Nickel Subsulfide, Nickel

  Updated version

  http://cancerres.aacrjournals.org/content/55/22/5251

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/55/22/5251To request permission to re-use all or part of this article, use this link

on March 29, 2019. © 1995 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from