Genetic susceptibility to nickel-induced acute lung injury

Genetic susceptibility to nickel-induced acute lung injury

Daniel R. Prows a,b,*, Susan A. McDowell a, Bruce J. Aronow c,George D. Leikauf a,d,e

a Department of Environmental Health, University of Cincinnati, Cincinnati, OH 45267-0056, USAb Division of Human Genetics, Children�s Hospital Medical Center, 3333 Burnet Ave., Building Code R, MLC 7016,

Cincinnati, OH 45229-3039, USAc Division of Pediatric Informatics, Children�s Hospital Medical Center, 3333 Burnet Ave., Building Code R, MLC 7024,

Cincinnati, OH 45229-3039, USAd Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, OH 45267, USA

e Department of Medicine, University of Cincinnati, Cincinnati, OH 45267, USA

Received 30 August 2001; received in revised form 15 July 2002; accepted 31 August 2002

Abstract

Human exposure to insoluble and soluble nickel compounds is extensive. Besides wide usage in many industries,

nickel compounds are contained in cigarette smoke and, in low levels, in ambient particulate matter. Soluble nickel

particulate, especially nickel sulfate (NiSO4), has been associated with acute lung injury. To begin identifying genes

controlling susceptibility to NiSO4, mean survival times (MSTs) of eight inbred mouse strains were determined after

aerosol exposure. Whereas A/J (A) mice were sensitive, C57BL/6J (B6) mice survived nearly twice as long (resistant).

Their offspring were similarly resistant, demonstrating heritability as a dominant trait. Quantitative trait locus (QTL)

analysis of backcross mice generated from these strains identified a region on chromosome 6 significantly linked to

survival time. Regions on chromosomes 1 and 12 were suggestive of linkage and regions on chromosomes 8, 9, and 16

contributed to the response. Haplotype analysis demonstrated that QTLs on chromosomes 6, 9, 12, and 16 could

explain the MST difference between the parental strains. To complement QTL analysis results, cDNA microarray

analysis was assessed following NiSO4 exposure of A and B6 mice. Significant expression changes were identified in one

or both strains for >100 known genes. Closer evaluation of these changes revealed a temporal pattern of increased cell

proliferation, extracellular matrix repair, hypoxia, and oxidative stress, followed by diminished surfactant proteins.

Certain expressed sequence tags clustered with known genes, suggesting possible co-regulation and novel roles in

pulmonary injury. Together, results from QTL and microarray analyses of nickel-induced acute lung injury survival

allowed us to generate a short list of candidate genes.

� 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Candidate gene; Haplotype analysis; Microarray; Particulate; QTL analysis; Survival

1. Introduction

Particulate matter (PM) levels associated with respi-

ratory morbidity and mortality are low compared to the

existing scientific literature, suggesting that individual

susceptibility differences may play a role in response.

Clinical studies demonstrated that individuals vary in

bronchoconstriction induced by ozone (O3), a common

respiratory irritant (McDonnell et al., 1985). Inbred

mouse strains also vary in sensitivity to O3-induced re-

spiratory effects, including acute lung injury (Stokinger,

1957; Goldstein et al., 1973; Ichinose et al., 1982;

Prows et al., 1997, 1999) and O3-induced increases in

Chemosphere 51 (2003) 1139–1148

www.elsevier.com/locate/chemosphere

*Corresponding author. Tel.: +1-513-636-5440; fax: +1-513-

636-4373.

E-mail address: [email protected] (D.R. Prows).

0045-6535/03/$ - see front matter � 2003 Elsevier Science Ltd. All rights reserved.

doi:10.1016/S0045-6535(02)00710-5

neutrophils or total proteins retrieved from bronchoal-

veolar lavage (BAL) fluid (Kleeberger et al., 1990,

1993a,b, 1997a). These studies indicated not only that

survival of O3-induced acute lung injury is controlled by

multiple genetic loci (i.e. polygenic), but also that the

major genes controlling susceptibility to acute lung in-

jury survival differ from those controlling neutrophil

influx or BAL total proteins.

Besides O3, other environmental and surrogate en-

vironmental pollutants have been shown to induce dif-

ferential responses in mice and rats. For example, inbred

mice show susceptibility differences to NO2-induced

pulmonary inflammation (Holroyd et al., 1997; Klee-

berger et al., 1997b) and to acid-coated particle-induced

macrophage phagocytosis (Ohtsuka et al., 2000). Recent

studies of residual oil fly ash (ROFA), a PM surrogate,

demonstrated that the water-soluble metals (e.g., vana-

dium, chromium, nickel, and iron) contained in ROFA

may be responsible for the observed acute lung injury

(Costa and Dreher, 1997; Dreher et al., 1997; Dye et al.,

1997). In vitro studies reported that the active compo-

nents of ROFA were vanadium, chromium, and nickel;

vanadium was the most active (Pritchard et al., 1996;

Kodavanti et al., 1998a,b). On the other hand, in vivo

studies demonstrated that nickel was the most biologi-

cally active of the metals found in ROFA (Pritchard

et al., 1996; Costa and Dreher, 1997; Kodavanti et al.,

1998a,b). Additional in vivo studies with intratracheal

instillation of ROFA found differences in rat strain

sensitivity, suggesting genetic susceptibility may play a

role in individual responsiveness to inhaled PM (Ko-

davanti et al., 1997).

Acute lung injury is characterized by a deficit in gas

exchange, owing to macrophage activation, epithelial

and endothelial disruption, and surfactant protein (SP)

dysfunction (Lewis and Jobe, 1993; Levy et al., 1995).

Because numerous insults can induce acute lung in-

jury, studying additional agonists could reveal common

mechanisms likely to involve pathways that control

macrophage activation, epithelial or endothelial injury,

and oxidative stress (Pryor et al., 1990; Stohs and Bag-

chi, 1995; Pritchard et al., 1996). For example, insolu-

ble ultrafine particulate, such as those generated

from polytetrafluoroethylene (PTFE), can induce acute

lung injury (Pryor et al., 1990; Johnston et al., 1996;

Oberd€oorster et al., 1998). In fact, we found a similar

strain phenotype pattern for acute lung injury survival in

mice following exposure to PTFE (Wesselkamper et al.,

2000) as seen with O3 (Prows et al., 1997). In both cases,

A/J (A) mice were sensitive and C57BL/6J (B6) mice

were relatively resistant to the induced acute lung injury.

Certain transition metals also can induce acute lung

injury. Of the transition metals enriched in the fine

fraction of ambient PM and the workplace, nickel

compounds can be especially harmful (NIOSH, 1977;

IARC, 1990; NTP, 1996). Nickel occurs primarily in

soluble (e.g., sulfate, chloride and acetate) and insoluble

(e.g., oxide and elemental nickel) forms. Nickel enters

the environment via many sources (Table 1), primarily

through high temperature combustion, electroplating,

and smelting processes (Senior and Flagan, 1982; Mil-

ford and Davidson, 1987; IARC, 1990; NTP, 1996). The

fraction of the population exposed to nickel in the en-

vironment is significant (Table 2), and still others are

exposed in the workplace (Table 3). Besides ambient and

occupational exposures, nickel (0.2–0.51 lg/m3) is a

component of mainstream cigarette smoke in concen-

trations greater than other metal ions, such as copper,

cadmium, and iron (0.19, 0.07–0.350, and 0.042 lg/ciga-rette, respectively) (IARC, 1986).

Previous inhalation exposures of rats and mice to

NiSO4 (Benson et al., 1988; Dunnick et al., 1988; Benson

et al., 1995; Dunnick et al., 1995; NTP, 1996) noted

acute and chronic respiratory effects. Because of the

prevalence of nickel exposures and the link to respira-

tory morbidity and mortality in laboratory animals, we

initiated studies using nickel concentrations at or near

the current occupational standard (i.e. TLV ¼ 100 lgNi/m3) to determine the effects of NiSO4 on different

strains of inbred mice. At these concentrations, NiSO4

produced an acute lung injury in inbred strains of mice,

ultimately resulting in death due to endothelial disrup-

Table 1

Sources of nickel exposure

Coal- and oil-fired power plants

Diesel-powered engines

Mining and refineries

Nickel electroplating

Waste incinerators

Table 2

Environmental exposures of nickel

Typical ambient nickel exposures

� Rural (background) levels:

0.0001–0.078 lg/m3

Milford and

Davidson (1987)

� Urban areas or downwind of

point source: 0.38–0.73 lg/m3

Schroeder et al.

(1987)

Chan and Lusis

(1986)

� Downwind of nickel mining

operations: 2.3–6.1 lg/m3

Dobrin and

Potvin (1992)

Brecher et al. (1989)

Exposures as high as 8–15.8 lg/m3 NTP (2000)

About 160 million US people live

within 12.5 miles of nickel source

NTP (2000)

Cigarette smoke: 0.2–0.51 lg/ciga-rette and up to 3 lg/cigarette

IARC (1986)

OSH (1982)

Positive skin test for nickel:

>15% of population

Mattila et al. (2001)

Sun (1987)

1140 D.R. Prows et al. / Chemosphere 51 (2003) 1139–1148

tion and hemorrhagic pulmonary edema (Wesselkamper

et al., 2000). This acute lung injury was similar to that

seen with O3- and PTFE-induced lung injuries. An im-

portant distinction, however, was that the NiSO4-

induced lung injury progressed much slower than that

caused by O3 or PTFE (Wesselkamper et al., 2000)––a

timeframe that more closely resembles the human con-

dition. Given that (1) little is known currently about

susceptibility differences to fine PM; (2) NiSO4 can be a

frequent component of environmental and occupation

air; and (3) NiSO4 can induce an acute lung injury with

a progression approximating that seen clinically, we

sought to determine a mouse model to identify the ge-

netic factors controlling survival differences to nickel-

induced acute lung injury. Once established, this mouse

model was utilized in quantitative trait locus (QTL),

haplotype, and cDNA microarray analyses to investi-

gate the genetic determinants of NiSO4-induced acute

lung injury.

2. Mouse model

The first step to determine a mouse model for genetic

analysis of nickel-induced acute lung injury was to

identify two strains of mice with significantly different

mean survival times (MSTs) to NiSO4 inhalation (i.e.

polar-responding strains). Eight commonly used inbred

mouse strains were obtained from Jackson Laboratory

(Bar Harbor, ME) and exposed continuously to 150 lgNi/m3 (mass median aerodynamic diameter (MMAD)¼0.22 lm, geometric standard deviation ðrgÞ ¼ 1:85)

generated from a solution of NiSO4 � 6H2O (Sigma, St.

Louis, MO). The strain phenotype pattern for survival is

displayed in Fig. 1. Similar to results from O3 and PTFE

exposures, the A strain was the most sensitive (MST ¼68� 4 h) and the B6 strain most resistant (MST ¼133� 5 h) to nickel-induced acute lung injury survival

(Wesselkamper et al., 2000). Also like O3 and PTFE

exposures, first generation progeny derived from the A

and B6 strains (B6AF1, MST ¼ 136� 4 h) demon-

strated that the resistance phenotype was inherited as a

dominant trait. Surprisingly, the sensitive A strain

demonstrated 20% mortality (2/10) in 15 lg Ni/m3 and

little difference in MSTs were noted among concentra-

tions P70 lg Ni/m3 (Wesselkamper et al., 2000). Using

the polar-responding A and B6 strains as a model to

identify the regions linked to this phenotype, we gener-

ated 307 backcross mice for QTL analysis. Combining

all exposed control A (n ¼ 52) and B6 (n ¼ 54) mice the

MSTs were refined to 67� 3 and 120� 3 h, respectively

(Prows and Leikauf, 2001).

3. QTL analysis

To initially identify possible QTLs influencing sur-

vival to nickel-induced lung injury, 77 microsatellite

markers were typed for the 55 most sensitive (survival

times 6 66 h) and 54 most resistant (survival times

P 112 h) backcross mice (representing the 109 pheno-

typic extreme-responders) (Prows and Leikauf, 2001).

Results were analyzed with MAPMAKER/QTL and the

theoretical levels for significant (lod score P 3.3) and

Table 3

Nickel concentrations in the workplace

Over 2000 Ni work sites¼ three times (Crþ Cdþ Co) sites Leikauf et al. (1995)

Asthma (associated with ambient levels >30 lg/m3) Bright et al. (1997)

Davies (1986)

Dolovich et al. (1984)

Malo et al. (1982)

Chronic obstructive pulmonary disease Nemery (1990)

Cornell and Landis (1984)

Polednak (1981)

Occupational nickel levels (estimated 1.5 million workers exposed in US)

Electroplating : 2–170 lg=m3

Welding : 10–1000 lg=m3

Battery manufacturing : 20–1910 lg=m3

Refining : 10–5000 lg=m3

9>>=>>;

Haber et al. (2000)

Mastromatteo (1986)

Warner (1984)

Threshold limit values (TLVs) for nickel compounds

Soluble : 100 lg=m3

Metallic nickel : 1000 lg=m3

Insoluble : 1000 lg=m3

9=;

ACGIH (2001)

D.R. Prows et al. / Chemosphere 51 (2003) 1139–1148 1141

suggestive (lod scoreP1.9) linkage proposed by Lander

and Kruglyak (1995) for a backcross analysis were used

to identify potential QTLs in the extreme responding

cohort. (Note: The lod score represents a log of the odds

ratio, which is the likelihood of linkage divided by the

likelihood of no linkage.) Regions reaching suggestive

linkage were identified on chromosomes 1, 6, 8, 9, 12

and 16, with lod scores for these six putative QTLs

ranging from 2.1 to 2.8. Assuming independent loci,

these QTLs explain 62% of the genetic variance in the

phenotypic extreme cohort.

To validate these QTLs in the total backcross popu-

lation, all 307 mice were typed for the original 77 mi-

crosatellite markers, with an additional 32 markers

located in and around the six putative QTL intervals.

Using QTL Cartographer analysis of the total backcross

cohort, significant (a ¼ 0:05) and suggestive (a ¼ 0:1)linkages were established at empirical levels determined

by 10 000 permutations of the original data set (Basten

et al., 1994, 1997). Experiment-wise threshold levels for

this data set were established at a lod scoreP 2.6 for

significant linkage and P2.3 for suggestive linkage. The

major findings of this analysis are displayed in Fig. 2. The

QTL on chromosome 6 was significantly linked, reaching

a peak lod score of 3.0 at D6Mit183. This QTL was

designated Aliq4 (for Acute lung injury qtl-4). (Note:

QTLs Aliq1-3 were previously named for loci linked to

O3-induced acute lung injury (see Prows et al., 1997)).

Suggestive linkages were identified on chromosome 1 (4-

cM distal to D1Mit213, lod score 2.5) and chromosome

12 (D12Mit185 and D12Mit112 both with lod scores of

2.3). The QTLs on chromosome 8 (peak at 6-cM distal to

D8Mit65, lod score 2.2), chromosome 9 (D9Mit227, lod

score 1.6) and chromosome 16 (D16Mit152, lod score

1.6), initially identified as suggestive loci in the pheno-

typic extreme backcrosses, did not reach the threshold

for experiment-wise suggestive linkage in the total back-

cross population. Genes mapping near and within these

six putative QTL intervals were considered positional

candidate genes for controlling some part of the response

to nickel sulfate inhalation.

4. Haplotype analysis

To determine the contribution of each QTL and QTL

combination to the overall phenotype, MSTs of mice

with a sensitive haplotype (i.e. sensitive alleles at the peak

marker of putative QTLs) were compared to MSTs of

mice with a resistant haplotype (Fig. 3; Prows and Lei-

kauf, 2001). For each backcross, only a homozygous A

(AA) or heterozygous (H) genotype could be obtained

for microsatellite marker typings. Microsatellite marker

D6Mit183 had the greatest difference in MST between

groups of mice containing either the AA or H genotype;

mice heterozygous at that locus survived an average of 12

h longer than AA mice. Different haplotype combina-

tions at two QTLs showed the greatest MST difference

for mice heterozygous for QTLs on chromosomes 1 and

6, with H–H mice surviving an average of 25 h longer

than mice AA–AA. For three QTLs, mice heterozygous

for markers representing chromosomes 6, 12, and 16 (H–

H–H) had a MST 40 h longer than mice AA at these

markers. The best agreement between phenotype and

genotype for four QTLs was noted with the haplotype

H–H–H–AA for markers on chromosomes 6, 12, 16, and

9, results that directly correlated with QTL results. Mice

with this haplotype survived an average of 52 h longer

than AA–AA–AA–H mice. Interesting, this difference in

MST correlated with the difference in MST between the

A and B6 parental strains. Analysis of the different

haplotypes for five QTLs showed a MST difference of 75

h for mice H–H–H–H–AA at chromosomes 1, 6, 12, 16,

and 9, respectively, compared to mice that were AA–

AA–AA–AA–H for these markers (Fig. 3). This in-

creased difference in MSTs from that of the parental

strains suggests that a resistance gene is contained in the

A strain, which is sensitive in the B6 strain––a finding

consistent with the opposing QTL on chromosome 9.

MST was not extended in mice carrying the resistance

alleles for the QTL on chromosome 8, making its im-

portance in the overall phenotype equivocal.

5. Microarray analysis

To complement QTL analysis we performed micro-

array analysis of 8734 sequence-verified murine cDNAs

Fig. 1. Survival times of eight inbred strains of mice during

continuous exposure to fine nickel sulfate (NiSO4). Mice were

exposed continuously to 150� 15 lg Ni/m3 (0.2 lm MMAD,

rg ¼ 1:9) and time of death recorded. White Bar: The mean

response for A/J mice; Gray Bars: Other sensitive and inter-

mediate strain responses [CBA/J, SPRET/Ei (SPRET), FVB/

NJ, DBA/2J, AKR/J, and C3H/HeJ]; Black Bar: The mean

response of resistant C57BL/6J (B6) mice. Values are MSTs�SE (n ¼ 6–27 mice/strain). Adapted from Wesselkamper et al.

(2000).

1142 D.R. Prows et al. / Chemosphere 51 (2003) 1139–1148

to assess gene expression differences between A and B6

mice following 3, 8, 24, and 48 h of NiSO4 exposure.

Details of this analysis for the B6 strain (at these ex-

posure times, as well as for 96 h) have been reported

(McDowell et al., 2000). To extend the initial analysis

of the B6 mice, we performed an analysis of gene ex-

pression in the lungs of A strain mice. Polyadenylated

mRNAs from three mice at 0 (control), 3, 8, 24, or 48

h exposure were pooled and used to generate target

cDNAs by reverse transcription with fluorescent-labe-

led random 9-mers. During reverse transcription, cDNAs

deriving from mRNA of control mice were fluorescent-

labeled with Cy5 (Met-5A) dUTP and cDNAs deriving

from mRNA of exposed mice were fluorescent-labeled

with Cy3 (MSTO-211H) dUTP. Hybridizations and

fluorescence measurements were conducted by Incyte

Pharmaceuticals (Freemont, CA). The ratio of total

Cy3 signal to the total Cy5 signal (balance coefficient)

was applied to normalize each of the eight microarray

slides used in this analysis. Multiplying the differential

fluorescence level (ratio of Cy5:Cy3 signal when Cy5 >Cy3, and ratio of Cy3:Cy5 signal when Cy5 < Cy3) of

each cDNA by the balance coefficient derived for each

microarray slide produced a balanced differential ex-

pression (BDE) ratio for each cDNA. Because our

analysis involves only one microarray (a pooled sample

of three mice) at each time for each strain, these pre-

liminary findings should be viewed with caution.

To identify the cDNAs (genes) with the greatest

difference between the strains, the acceptance stringency

was set at a BDE ratioP j2j for at least one time in

one or both strains. Using this threshold, 135 different

Fig. 2. QTL analysis results for chromosomes 1, 6, 8, and 12 from the total backcross population (n ¼ 307). Open circles represent

MIT microsatellite marker positions. Solid (at 2.58) and dashed (at 2.30) lines denote significant or suggestive linkage, respectively, as

determined empirically by 10 000 permutations of the data set using QTL Cartographer (Basten et al., 1997). The peak on chromosome

8 was below suggestive linkage. The major QTL (Aliq4) peaked at marker D6Mit183 on chromosome 6. Adapted from Prows and

Leikauf (2001).

D.R. Prows et al. / Chemosphere 51 (2003) 1139–1148 1143

cDNAs (107 for known genes and annotated expressed

sequence tags, ESTs, and 28 nonannotated ESTs) were

identified with significant expression changes in one or

both strains. Of the 107 known genes and annotated

ESTs, the expression of 38 changed to the same extent

in both strains, 18 expression changes were significant

in A strain mice, and 51 were significant in B6 mice.

Among the expression changes, four groups could be

identified. First, are cDNAs with expression signifi-

cantly changed in both strains, but higher in A mice;

these include genes increased in A more than B6 mice

(e.g., Eph receptor A2) or decreased in B6 more than A

mice (e.g., SP-B). Second, are cDNAs whose expression

was significantly changed in both strains, but the ex-

pression was higher in B6 mice; these include genes

with expression increased in B6 more than A mice (e.g.,

metallothionein-1) or decreased in A more than B6

mice (e.g., Clara cell secretory protein). The last two

groups included those cDNAs whose expression was

significantly increased or decreased only in A mice

(e.g., calgranulin A and hemoglobin-a adult chain-1,

respectively) or only in B6 mice (e.g., metal-response

element (MRE) DNA binding transcription factor-2,

and platelet activating factor acetylhydrolase 1B a,respectively). Closer scrutiny of these group members

identified 15 functional candidate genes (Table 4) that

demonstrated a BDE ratioP j2j between strains for at

least one exposure time.

6. Combining QTL and microarray analysis

To gain further insight into the important genes as-

sociated with survival to nickel-induced acute lung in-

Table 4

Candidate genes for nickel-induced acute lung injurya

Name Abbreviation Locationb

Calgranulin A S100a8 3, 43.6 cM

Clara cell secretory protein (Uteroglobin) Utg 19(A)

Cytochrome P450 2f2 Cyp2f2 7, Syntenic

Enolase-3 Eno3 11, 42 cM

Galectin-3 (Lectin, galactose binding, soluble-3) Lgals3 14(C1)

Glyceraldehyde-3-phosphate dehydrogenase Gapd 6, 56 cM

Hemoglobin a, adult chain-1 Hba-a1 11, 16 cM

Hemolytic complement Hc 2, 23.5 cM

Keratin complex 2, basic gene-7 (EST) Krt2-7 11, 58.7 cM

Metallothionein-1c Mt1 8, 45 cM

Metal response element binding transcription factor-2 Mtf2 Unknown

N-myc downstream regulator Ndr1 15, Syntenic

Surfactant protein-B Sftpb 6, 31 cM

Surfactant protein-C Sftpc 14, 32.5 cM

Thioredoxin reductase-1 Txnrd1 10, Syntenic

aAll genes listed were derived from microarray analysis of A and B6 strains and had a BDE ratioP j2j in at least one strain and one

exposure time.bGenome location is given as chromosome, centimorgan (cM) position from the centromere (from Jackson Laboratories webpage,

www.jax.org).c Text in bold represent genes located within a region linked by QTL analysis and with a BDE ratioP j2j between the A and B6

strains.

Fig. 3. Survival time differences of backcross mice with resis-

tance or sensitivity haplotypes at microsatellite markers repre-

senting the putative QTLs. The highlighted pair represents

survival times of A/J (A) and C57BL/6J (B) parental strains.

White bars¼ backcross mice with AA genotype (A); black

bars¼backcross mice with AB or BA genotype (H), or BB

genotype (B) for C57BL/6J parental controls. Number above

each pair represents the survival time difference (in hours) be-

tween backcross mice with the designated haplotypes. Values

are the MSTs� SEs. All comparisons of sensitivity (white bars)

versus resistance (black bars) haplotypes were significant (P <

0:05, t-test). Adapted from Prows and Leikauf (2001).

1144 D.R. Prows et al. / Chemosphere 51 (2003) 1139–1148

jury, we compared the chromosomal locations identified

by QTL analysis with the genes identified through ex-

pression changes in the cDNA microarray analysis.

Among the 15 functional candidate genes (i.e. those

genes showing a BDE ratioP j2j between strains), two

genes––metallothionein-1 (Mt1) on chromosome 8 and

SP-B (Sftpb) on chromosome 6––map to QTL intervals

linked to nickel-induced acute lung injury survival

(Prows and Leikauf, 2001).

In addition, several other genes showing a significant

expression change in one or both mouse strains map

within or near chromosomal regions linked to the phe-

notype, including procollagen type III, a-1 (21 cM on

chromosome 1), glutathione reductase-1 (18 cM on

chromosome 8), esterase-1 (43 cM on chromosome 8),

heme oxygenase-1 (C1 band on chromosome 8), and

nuclear receptor coactivator-1 (bands A2–A3 on chro-

mosome 12). The other positional candidate genes sug-

gested previously (Prows and Leikauf, 2001) were either

not on the microarray or their expression levels did not

change to the established thresholds during the expo-

sures.

7. Summary and discussion

We have outlined the methods used to generate a

short list of functional and positional candidate genes

for nickel-induced acute lung injury survival. Initially, a

mouse model was determined, which represented two

strains of inbred mice (A and B6) that responded sig-

nificantly different to a continuous NiSO4 inhalation

exposure. Next, after establishing that survival was a

polygenic trait and that resistance was dominantly in-

herited, backcross mice were generated for genetic

studies. Subsequent to exposure and genotyping of 105

microsatellite markers distributed throughout the ge-

nome, QTL analysis of 307 backcross mice identified a

region on chromosome 6 (proposed as Aliq4) signifi-

cantly linked to survival. Several interesting positional

candidate genes map to this interval, including Sftpb and

transforming growth factor (TGF)-a (Tgfa). In addition

to the most significant QTL on chromosome 6, several

modifier loci on chromosomes 1, 9, 12, and 16 were re-

vealed through haplotype analysis to influence the phe-

notype. One further peak on chromosome 8 approached

the threshold for suggestive linkage in the QTL analysis

(i.e. it had a lod score of 2.2 where the suggestive level

was 2.3). Next, microarray analysis was performed to

assess gene expression changes at four times throughout

the nickel exposure. Several functional candidate genes

were determined from this analysis, based on the dif-

ferential expressions between the A and B6 strains. Re-

sults from QTL, haplotype, and microarray analyses are

ambiguous for the QTL on chromosome 8, so its im-

portance is still uncertain. However, as identified by

microarray analysis, several interesting positional can-

didates for acute lung injury map to this region (e.g.,

esterase-1, glutathione reductase-1, heme oxygenase-1,

and metallothionein-1), and displayed significant ex-

pression changes in one or both strains. We then com-

bined QTL and microarray results to create a short list

of candidate genes for further studies.

Two main strategies can be used to extend these re-

sults. First, to refine the QTL intervals to regions that

will allow physical mapping, congenic lines of mice can

be constructed for each QTL. Once constructed, these

strains can also be bred further to generate multi-con-

genic lines to evaluate additive and epistatic effects of the

QTLs to the overall phenotype. Haplotype analysis

suggested that a multi-congenic line with resistance al-

leles for QTLs on chromosomes 6, 12, and 16 on the

sensitive A background (which would already have the

resistance alleles for the QTL on chromosome 9) could

result in a phenotype similar to the resistant B6 parental

strain. Haplotype analysis also suggested that fixing the

sensitive alleles for the QTL on chromosome 1 in these

multi-congenics would result in a MST significantly

longer than the B6 parent would. Likewise, placing the

sensitive alleles for QTLs on chromosomes 6, 12 and 16

onto the B6 background (which also has sensitive alleles

for chromosome 9 QTL) would result in a phenotype

similar to the sensitive A strain parent.

Another method that can be undertaken concur-

rently with the construction of congenic lines is a po-

sitional candidate-gene approach (Collins, 1995). As

mentioned above, Sftpb and Tgfa are two genes located

within the interval spanning a 1-lod unit decrease on

either side of the Aliq4 peak (D6Mit183) on chromo-

some 6. Microarray analysis found SP-B gene expression

to decrease significantly with continued nickel exposure

(McDowell et al., 2000) and SP-B has been implicated as

an important factor in acute lung injury (Luce, 1998).

TGF-a is a polypeptide member of a protein family that

includes epidermal growth factor (EGF) and other li-

gands of the EGF receptor (Derynck, 1988). Strong

physiological evidence of a role for TGF-a in acute lung

injury resistance was provided when mice overexpressing

human TGF-a in the lung demonstrated a significant

increase in MST compared to littermate controls fol-

lowing exposure to ultrafine PTFE particles (Hardie

et al., 1999).

In theory, any of the expression differences deter-

mined on the microarrays could represent potential

functional candidate genes that control at least part of

the disparity in phenotypic response. By combining mi-

croarray analysis with QTL analysis our goal was to

identify genes that reside within a putative QTL interval

and differ in gene expression between the two strains,

thereby significantly decreasing the number of gene

candidates. This combined approach reduced the num-

ber of genes to two––Sftpb andMt1. A third gene, Tgfa,

D.R. Prows et al. / Chemosphere 51 (2003) 1139–1148 1145

was not on the microarray but demonstrated a physio-

logical importance to the phenotype. It is unknown,

however, whether these genes are specific for nickel-

induced acute lung injury, or whether they may be

related to a generalized agent-induced lung response.

In addition, many potential and obvious pitfalls exist

with this combined strategy. First, other potential can-

didates likely include genes with lesser differences be-

tween the strains, but were not detected by the more

stringent thresholds used in the microarray study. Sec-

ond, gene expression may not correlate with protein

function, such that a gene with similar expression in two

strains may lead to significantly different protein func-

tions. Third, many potential candidate genes were not

included on the available microarray; so many important

genes were not screened. Fourth, many of the cDNAs on

the microarray are yet to be mapped or annotated in the

mouse. Regardless of these shortcomings, the indepen-

dent and combined approaches presented herein have

highlighted many functional and positional candidate

genes, which can be further evaluated for their possible

role(s) in nickel-induced acute lung injury survival.

Acknowledgements

This study was supported by the NHLBI (HL65213

and HL65612), NIEHS (ES10562 and ES06096), and the

Health Effects Institute (HEI), an organization jointly

funded by the US Environmental Protection Agency

(EPA), Assistance Agreement X-812059, and the auto-

motive manufacturers. The contents of this article do

not necessarily reflect the views of the HEI or the poli-

cies of the US EPA or automotive manufacturers.

References

ACGIH, American Conference of Governmental Industrial

Hygienists, 2001. Threshold limit values for chemical

substances and physical agents and biological indices.

ISBN. 1-882417-40-2.

Basten, C.J., Weir, B.S., Zeng, Z.-B., 1994. Zmap––A QTL

Cartographer. In: Smith, C., Gavora, J.S., Benkel, B.,

Chesnais, J., Fairfull, W., Gibson, J.P., Kennedy, B.W.,

Burnside, E.B. (Eds.), 5th World Conference on Genetics

Applied to Livestock Production: Computing Strategies and

Software, Ontario, Canada 22, pp. 65–66.

Basten, C.J., Weir, B.S., Zeng, Z.-B., 1997. QTL Cartographer:

A Reference Manual and Tutorial for QTL Mapping.

Department of Statistics, NC State University, Raleigh,

NC.

Benson, J.M., Burt, D.G., Carpenter, R.L., Eidson, A.F., Hahn,

F.F., Haley, P.J., Hanson, R.L., Hobbs, C.H., Pickrell, J.A.,

Dunnick, J.K., 1988. Comparative inhalation toxicity of

nickel sulfate to F344/N rats and B6C3F1 mice exposed for

twelve days. Fundam. Appl. Toxicol. 10, 164–178.

Benson, J.M., Cheng, Y.S., Eidson, A.F., Hahn, F.F., Hender-

son, R.F., Pickrell, J.A., 1995. Pulmonary toxicity of nickel

subsulfide in F344/N rats exposed for 1–22 days. Toxicology

103, 9–22.

Brecher, R.W., Austen, M., Light, H.E., Stepein, E., 1989.

Contract report for the Environmental Substances Division,

Environmental Health Center, Health and Welfare Canada,

Ottawa, Ontario. Eco Logic, Inc.

Bright, P., Burge, P.S., O�Hickey, S.P., Gannon, P.F., Robert-

son, A.S., Boran, A., 1997. Occupational asthma due to

chrome and nickel electroplating. Thorax 52, 28–32.

Chan, W.H., Lusis, M.A., 1986. Smelting operations and trace

metals in air and precipitation in the Sudbury Basin. Adv.

Environ. Sci. Technol. 17, 113–143.

Collins, F.S., 1995. Positional cloning moves from perditional

to traditional. Nat. Genet. 9, 347–350.

Cornell, R.G., Landis, J.R., 1984. Mortality patterns among

nickel/chromium alloy foundry workers. IARC Sci. Publ.

53, 87–93.

Costa, D.L., Dreher, K.L., 1997. Bioavailable transition metals

in particulate matter mediate cardiopulmonary injury in

healthy and compromised animal models. Environ. Health

Perspect. 105, 1053–1060.

Davies, J.E., 1986. Occupational asthma caused by nickel salts.

J. Soc. Occup. Med. 36, 29–31.

Derynck, R., 1988. Transforming growth factor alpha. Cell 54,

593–595.

Dobrin, D.J., Potvin, R., 1992. Air Quality Monitoring Studies

in the Sudbury Area: 1978 to 1988. Ontario Ministry of the

Environment, Technical Assessment Section, Northeastern

Region, Ontario.

Dolovich, J., Evans, S.L., Nieboer, E., 1984. Occupational

asthma from nickel sensitivity: I. Human serum albumin in

the antigenic determinant. Br. J. Ind. Med. 41, 51–55.

Dreher, K.L., Jaskot, R.H., Lehmann, J.R., Richards, J.H.,

McGee, J.K., Ghio, A.J., Costa, D.L., 1997. Soluble tran-

sition metals mediate residual oil fly ash induced acute lung

injury. J. Toxicol. Environ. Health 50, 285–305.

Dunnick, J.K., Benson, J.M., Hobbs, C.H., Hahn, F.F., Cheng,

Y.S., Eidson, A.F., 1988. Comparative toxicity of nickel

oxide, nickel sulfate hexahydrate, and nickel subsulfide after

12 days of inhalation exposure to F344/N rats and B6C3F1

mice. Toxicology 50, 145–156.

Dunnick, J.K., Elwell, M.R., Radovsky, A.E., Benson, J.M.,

Hahn, F.F., Nikula, K.J., Barr, E.B., Hobbs, C.H., 1995.

Comparative carcinogenic effects of nickel subsulfide, nickel

oxide, or nickel sulfate hexahydrate chronic exposures in the

lung. Cancer Res. 55, 5251–5256.

Dye, J.A., Adler, K.B., Richards, J.H., Dreher, K.L., 1997.

Epithelial injury induced by exposure to residual oil fly-ash

particles: role of reactive oxygen species? Am. J. Respir.

Cell. Mol. Biol. 17, 625–633.

Goldstein, B.D., Lai, L.Y., Ross, S.R., Cuzzi-Spada, R., 1973.

Susceptibility of inbred mouse strains to ozone. Arch.

Environ. Health 27, 412–413.

Haber, L.T., Erdreicht, L., Diamond, G.L., Maier, A.M.,

Ratney, R., Zhao, Q., Dourson, M.L., 2000. Hazard

identification and dose response of inhaled nickel-soluble

salts. Regul. Toxicol. Pharmacol. 31, 210–230.

Hardie, W.D., Prows, D.R., Leikauf, G.D., Korfhagen, T.R.,

1999. Attenuation of acute lung injury in transgenic mice

1146 D.R. Prows et al. / Chemosphere 51 (2003) 1139–1148

expressing human transforming growth factor-alpha. Am. J.

Physiol. 277, L1045–L1050.

Holroyd, K.J., Eleff, S.M., Zhang, L.Y., Jakab, G.J., Kleeber-

ger, S.R., 1997. Genetic modeling of susceptibility to nitro-

gen dioxide-induced lung injury in mice. Am. J. Physiol.

273, L595–L602.

IARC, International Agency for Research on Cancer, 1986.

Monographs on the Evaluation of Carcinogenic Risks to

Humans: Tobacco Smoking. IARC, Lyon, France.

IARC, International Agency for Research on Cancer, 1990.

Monographs on the Evaluation of Carcinogenic Risks to

Humans: Chromium, Nickel, and Welding. IARC, Lyon,

France.

Ichinose, T., Suzuki, A.K., Tsubone, H., Sagai, M., 1982.

Biochemical studies on strain differences of mice in the

susceptibility to nitrogen dioxide. Life Sci. 31, 1963–1972.

Johnston, C.J., Finkelstein, J.N., Gelein, R., Baggs, R.,

Oberd€oorster, G., 1996. Characterization of the early pul-

monary inflammatory response associated with PTFE fume

exposure. Toxicol. Appl. Pharmacol. 140, 154–163.

Kleeberger, S.R., Bassett, D.J., Jakab, G.J., Levitt, R.C., 1990.

A genetic model for evaluation of susceptibility to ozone-

induced inflammation. Am. J. Physiol. 258, L313–L320.

Kleeberger, S.R., Levitt, R.C., Zhang, L.Y., 1993a. Suscepti-

bility to ozone-induced inflammation. I. Genetic control of

the response to subacute exposure. Am. J. Physiol. Lung

Cell. Mol. Physiol. 264, L15–L20.

Kleeberger, S.R., Levitt, R.C., Zhang, L.Y., 1993b. Suscepti-

bility to ozone-induced inflammation. II. Separate loci

control responses to acute and subacute exposures. Am. J.

Physiol. Lung Cell. Mol. Physiol. 264, L21–L26.

Kleeberger, S.R., Levitt, R.C., Zhang, L.Y., Longphre, M.,

Harkema, J., Jedlicka, A., Eleff, S.M., DiSilvestre, D.,

Holroyd, K.J., 1997a. Linkage analysis of susceptibility to

ozone-induced lung inflammation in inbred mice. Nat.

Genet. 17, 475–478.

Kleeberger, S.R., Zhang, L.-Y., Jakob, G.J., 1997b. Differential

susceptibility to oxidant exposure in inbred strains of mice:

nitrogen dioxide versus ozone. Inh. Toxicol. 9, 601–621.

Kodavanti, U.P., Jaskot, R.H., Su, W.Y., Costa, D.L., Ghio,

A.J., Dreher, K.L., 1997. Genetic variability in combustion

particle-induced chronic lung injury. Am. J. Physiol. Lung

Cell. Mol. Physiol. 272, L521–L532.

Kodavanti, U.P., Hauser, R., Christiani, D.C., Meng, Z.H.,

McGee, J., Ledbetter, A., Richards, J., Costa, D.L., 1998a.

Pulmonary responses to oil fly ash particles in the rat differ

by virtue of their specific soluble metals. Toxicol. Sci. 43,

204–212.

Kodavanti, U.P., Meng, Z.H., Hauser, R., Christiani, D.C.,

Ledbetter, A., McGee, J., Richards, J., Costa, D.L., 1998b.

In vivo and in vitro correlates of particle-induced lung

injury: specific roles of bioavailable metals. In: Mohr, U.,

Dungworth, D.L., Brain, J.D., Driscoll, K.E., Grafstrom,

R.C., Harris, C.C. (Eds.), Relationships Between Respira-

tory Disease and Exposure to Air Pollution. ILSI Press,

Washington, DC.

Lander, E., Kruglyak, L., 1995. Genetic dissection of complex

traits: guidelines for interpreting and reporting linkage

results. Nat. Genet. 11, 241–247.

Leikauf, G.D., Kline, S., Albert, R.E., Baxter, C.S., Bernstein,

D.I., Buncher, C.R., 1995. Evaluation of a possible associ-

ation of urban air toxics and asthma. Environ. Health

Perspect. 103, 253–271.

Levy, P.C., Utell, M.J., Sickel, J.Z., Apostolakos, M.J., 1995.

The acute respiratory distress syndrome: current trends in

pathogenesis and management. Compr. Ther. 21, 438–444.

Lewis, J.F., Jobe, A.H., 1993. Surfactant and the adult

respiratory distress syndrome. Am. Rev. Respir. Dis. 147,

218–233.

Luce, J.M., 1998. Acute lung injury and the acute respiratory

distress syndrome. Crit. Care Med. 26, 369–376.

Malo, J.L., Cartier, A., Doepner, M., Nieboer, E., Evans, S.,

Dolovich, J., 1982. Occupational asthma caused by nickel

sulfate. J. Allergy Clin. Immunol. 69, 55–59.

Mastromatteo, E., 1986. Yant memorial lecture. Nickel. Am.

Ind. Hyg. Assoc. J. 47, 589–601.

Mattila, L., Kilpelainen, M., Terho, E.O., Koskenvuo, M.,

Helenius, H., Kalimo, K., 2001. Prevalence of nickel allergy

among Finnish university students in 1995. Contact Der-

matitis 44, 218–223.

McDonnell, W.F., Horstman, D.H., Abdul-Salaam, S., House,

D.E., 1985. Reproducibility of individual responses to

ozone exposure. Am. Rev. Respir. Dis. 131, 36–40.

McDowell, S.A., Gammon, K., Bachurski, C.J., Wiest, J.S.,

Leikauf, J.E., Prows, D.R., Leikauf, G.D., 2000. Differen-

tial gene expression in the initiation and progression of

nickel-induced acute lung injury. Am. J. Respir. Cell. Mol.

Biol. 23, 466–474.

Milford, J.B., Davidson, C.I., 1987. The sizes of particulate

sulfate and nitrate in the atmosphere––a review. J. Air Poll.

Control Assoc. 37, 125–134.

Nemery, B., 1990. Metal toxicity and the respiratory tract. Eur.

Respir. J. 3, 202–219.

NIOSH, National Institute for Safety and Health, 1977. Criteria

for a Recommended Standard: Occupational Exposure to

Inorganic Nickel. US Government Printing Office, Wash-

ington, DC, DHEW-NIOSH Document No. 77-164.

NTP, National Toxicology Program, 1996. Toxicology and

Carcinogenesis Studies of Nickel Sulfate in F344/N Rats

and B6C3F1 Mice (Inhalation Studies). (CAS No. 10101-97-

0) Research Triangle Park, NC, US Department of Health

and Human Services, Public Health Service, National

Institutes of Health, NTP-TRS No. 454.

NTP, National Toxicology Program, 2000. Ninth Summary

Report on Carcinogens. Research Triangle Park, NC, US

Department of Health and Human Services, Public Health

Services, National Institutes of Health. Available from

<http://ehis.niehs.nih.gov/roc/toc9.html>.

Oberd€oorster, G., Gelein, R., Johnston, C., Mercer, P., Corson,

N., Finkelstein, J.N., 1998. Ambient ultrafine particles:

inducers of acute lung injury? In: Mohr, U., Dungworth,

D.L., Brain, J.D., Driscoll, K.E., Grafstrom, R.C., Harris,

C.C. (Eds.), Relationships Between Respiratory Disease and

Exposure to Air Pollution. ILSI Press, Washington, DC.

Ohtsuka, Y., Clarke, R.W., Mitzner, W., Brunson, K., Jakab,

G.J., Kleeberger, S.R., 2000. Interstrain variation in murine

susceptibility to inhaled acid-coated particles. Am. J. Phys-

iol. Lung Cell. Mol. Physiol. 278, L469–L476.

OSH, Office on Smoking and Health, 1982. The Health

Consequences of Smoking and Cancer: A Report of the

Surgeon General. Department of Health and Human

Services (PHS), Publication No. 82-50179, Rockville, MD.

D.R. Prows et al. / Chemosphere 51 (2003) 1139–1148 1147

Polednak, A.P., 1981. Mortality among welders, including a

group exposed to nickel oxides. Arch. Environ. Health 36,

235–242.

Pritchard, R.J., Ghio, A.J., Lehman, J.R., Winsett, D.W.,

Tepper, J.S., Park, P., Gilmour, M.I., Dreher, K.L., Costa,

D.L., 1996. Oxidant generation and lung injury after

particulate air pollutant exposure increases with the con-

centrations of associated metals. Inh. Toxicol. 8, 457–477.

Prows, D.R., Leikauf, G.D., 2001. Quantitative trait analysis of

nickel-induced acute lung injury in mice. Am. J. Respir.

Cell. Mol. Biol. 24, 740–746.

Prows, D.R., Daly, M.J., Shertzer, H.G., Leikauf, G.D., 1999.

Ozone-induced acute lung injury: genetic analysis of F2 mice

generated from A/J and C57BL/6J strains. Am. J. Physiol.

Lung Cell. Mol. Physiol. 277, L372–L380.

Prows, D.R., Shertzer, H.G., Daly, M.J., Sidman, C.L.,

Leikauf, G.D., 1997. Genetic analysis of ozone-induced

acute lung injury in sensitive and resistant strains of mice.

Nat. Genet. 17, 471–474.

Pryor, W.A., Nuggehalli, S.K., Scherer Jr., K.V., Church, D.F.,

1990. An electron spin resonance study of the particles

produced in the pyrolysis of perfluoro polymers. Chem. Res.

Toxicol. 3, 2–7.

Schroeder, W.H., Dobson, M., Kane, D.M., Johnson, N.D.,

1987. Toxic trace elements associated with airborne partic-

ulate matter: a review. J. Air Poll. Control Assoc. 37, 1267–

1285.

Senior, C.L., Flagan, R.C., 1982. Ash vaporization and

condensation during combustion of a suspended coal

particle. Aerosol. Sci. Tech. 1, 371–383.

Stohs, S.J., Bagchi, D., 1995. Oxidative mechanisms in the

toxicity of metal ions. Free Radic. Biol. Med. 18, 321–336.

Stokinger, H.E., 1957. Evaluation of hazards of ozone and

oxides of nitrogen: factors modifying toxicity. Arch. Ind.

Health 15, 181–190.

Sun, C.C., 1987. Allergic contact dermatitis of the face from

contact with nickel and ammoniated mercury in spectacle

frames and skin-lightening creams. Contact Dermatitis 17,

306–309.

Warner, J.S., 1984. Occupational exposure to airborne nickel in

producing and using primary nickel products. IARC Sci.

Publ. 53, 419–437.

Wesselkamper, S.C., Prows, D.R., Biswas, P., Willeke, K.,

Bingham, E., Leikauf, G.D., 2000. Genetic susceptibility to

irritant-induced acute lung injury in mice. Am. J. Physiol.

Lung Cell. Mol. Physiol. 279, L575–L582.

1148 D.R. Prows et al. / Chemosphere 51 (2003) 1139–1148