Articles

The 5-Aminolevulinic Acid Dehydratase {ALAD) Polymorphism and Bone andBlood Lead Levels in Community-Exposed Men: The Normative Aging StudyHoward Hu,^^ Ming-Tsang Wu/ Yawen Cheng/ David Sparrow,^ Scott Weiss/ and Karl

"•Occupational Health Program, Department of Environmental Health, Harvard School of Public Health, Boston. Massachusetts, USA;^Channing Laboratory, Department of Medicine, Brigtiam and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA;^The Normative Aging Study, Department of Veterans Affairs, Boston, Massachusetts, USA; ''Department of Cancer Cell Biology.Harvard School of Public Health, Boston, Massachusetts, USA

Recent research has indicated that a polymorphic variant oF6'aminoleviilinic acid dehydratase{AIAD) may infltieiicc an individual's level of lead in bone and blood and, as a resuh. may alsoinfluence an individual's susceptibility to lead toxiciry. In ihis study, wt investigated whether this/4/../ /.) polymorphism is a.wociated with ahered levels of lead in bone and blood among " 26 mid-dle aged and elderly men who had community (nonoccupational) exposures to lead. We measuredlevels of blood and bone lead by graphite furnace atomic absorption spectroscopy and a K X-rayfluorescence (KXRF) instrument, respectively. We determined the AIAOMs\>\ polymorphism inexon 4 by a polynierase chain reaction restriction fragment length polymorphism (RFLPj. Of the726 subjects, 7 |1%I and 111 (15%) were, respectively, homozygous and heterozygous lor thevariant allelc. The mean (SD) of blood lead (micrograms per deciliter), cortical bone (tibia) lead(micrograms per gram), and trabecular bone (patella) lead (micrograms per gram) were 6.2 (4,1),22.1 (13.5), and 31,9 (19.5) in subjects who did not have the variant allele {ALAD J-I), and 5,7(4.2), 21.2 (10.9), and 30.4 (17.2) in the combined subjects who were either heterozygous orhomozygou.s for tbe variant allele {ALAD 1-2 and AIAD 2-2). In multivariate linear regressionmodels that controlled for age, education, smoking, alcohol ingestion, and vitamin D intake, theAIAD 1-1 genot)pe was associated with cortical bone lead levels that were 2.55 fig/g [95% confi-dence interval (Cl) 0.05-5-05] higher than those ofthe variant allele carriers. We found no signif-icant differences by genotype with respect to tead IeveLi in trabecular bone or blood. In stratifiedanalyses and a multivariate regression model thai tested for interaction, the relationship of trabec-ular bone lead to blood lead appeared to be significantly modified by (4Z-4O genotype, with vari-ant allele carriers having higher blood lead levels, but only when trabecular bone lead levelsexceeded 60 pg/g. These results suggest that the variant ALAD-2 allele modifies lead kinetics pos-sibly by decreasing lead uptake into cortical bone and increasing the mobilization of lead fromtrabecular bone. Key woreh. blood, bone, f)-aminolevulinic acid dehydrata.'ie, lead. Environ HealthPerspect 109:827-832 (2001]. [Online 13 August 2001|http://ehpnetLniehs.mh.gov/docs/2001/I09p827-832hu/abstract.hlml

Lead toxicity remains a critical environmen-cal health issue, given the widespread natureof lead exposure from both community andoccupacional exposures as well as the demon-stration of lead\s deleterious effect on multi-ple organ systems at ever lower levels ofexposure.

One important question in the study oflead toxicity char has only recently begunreceiving atretition involves understanding rhefactors that explain differences in symptomsamong individuals who have similar leadexposures. In addition, significant variationhas been observed in the relationship betweenbiologic markers of lead exposure and mea-sures of" organ dysfunction. Understandingthe nature ot this variation could al.so lead co amote profound understanding oF the mecha-nism of toxicity of lead.

A generic polymorphism in the fi-amino-levulinic acid dehydratase {AIAD) gene hasbeen suggested {1.2) to modify rhe kineticsand distribution of lead (and therefore itstoxicity). This gene codes for the seconden7yme in the biosynthetic pathway of heme{3,4). In 1981, Battistuzzi et al. (J) showed

that human ALAD protem is a polymorphicenzyme. Subsequently, Wetmur et al. {4)characreri7x'd the molecular nature of thispolymorphism, showing it to be caused by aG-to-C transversion in nucleoride 177. Thisproduces coding for lysine rather than aspar-ganine. Although initially Battistuzzi et al.(3) noted no detectable difference in the invitro activities ofthe variant enzymes {ALADJ-1. ALAD 1-2, or AIAD 2-2) in erythro-cytes, Bergdahl et al (5) found that the^ / J 4 Z ) - 2 subunit binds lead more dghtlythan does the ALAD-1 subunit.

Epidemiologic studies have suggestedthat among lead-exposed workers {6,7} andenvironmentally exposed children (6), indi-viduals with cither the ALAL') 7-2 or 2-2genotype had blood lead levels that were sig-nificantly higher than the blood lead levels ofindividuals with the ALAD 1-1 genotype.These findings suggest that the AlAD-2polypcptide binds lead more tightly andeffectively than ALAD-l. However, Smith etal. {8) did nor find any meaningful differ-ence in mean blood lead concentrationbetween y l i^O-^ carriers and those who

were homozygous for the ALAD-! aileleamong 688 members of a construction tradetinion. The relatively low blood lead levels ofthese union members [mean ± standarddeviation (SO): 7.8 ± 3.6 jig/dl-l led theauthors to suggest that the AIAD polymor-phism may influence lead kinetics, but onlyat relatively high levels of exposure. In stud-ies of lead smelter workers whose blood leadlevels ranged from 2()-.M) |.ig/dl., Bergdahl etal. {9) did not find any significant dilTerencesin bone and blood lead levels by genotypeamong 12.3 subjects, but Fleming et al, {10)found that lead workers with the ALAD 1-2/2-2 had significantly higher blood lead lev-els than those with AIAD I-l among 381subjects.

In this study, we investigated the impactof the .^/yJ/) polymorphism on lead levels inboth blood and bone among participants ofthe Normative Aging Stud}' (NAS) ( / / ) .This is a well-studied cohort of men, nowmiddle-aged and elderly, who have had gen-eral community (nonoccupational) expo-sures to lead. Among the advantages of thisstudy are the relatively large sample size (n =726), the availability of a host of oilier well-validaied demographic and lifestyle data, and

Adilrt-sa correspiuidi-dn.- lo It. ttii. CharmingLaboratory, 181 Lxingwood Ave., Boston, MA 0211 5USA. Telephone: (617) 52'i-2736. Fax: (6I7> 525-O.Wi2. F-mait: I low a rd.hii&"'ch an ning.harvard.edu

We gratefully acknowledge ttie research assistanceof S. Harcourt, R. Heldman, G. Barbclta. S.Oliveira, T. Luu, C. FIi-iscliat(cr, M. Barr. L.Hennessey, and S. Datta. D. Burger and F. Milderprovided technical a.ssistancc in ihe initial ph.isc ofour KXRF nicasurcmenis. J. McCoy provided edi-torial assiiiaiice. Finally, we are indebted, a.s always,to tht continued enthusiastic cooperation of iheparticipants in the Normative Aging Study.

Support tor this research was provided by NIEHSgrants EiS 05257-06At and t'42-h:S059'i7 I'rojcct 4.NIEHS Occupational and Environmentai HcalihC:cnti-r gram 2 I'. O ES00002. The Normative/^ing Study is siipportfd by the cooperative studiesprogram/ERIC, U.S. Departmcni of VeteransAffairs, and is a component tif" the MassachusettsVeterans Epidemiology Research and IntormationCenter (MAVlvRtC). Subjects were evaluated in theoiitpatieni Clinical Rese.irch C i-ntcr of IIK- Brighamand Womerrs Hospital widi support from NIHgrant NCRR CCRC MO1RR112W5. Iho KXRFinstrument used in this work was developed byABtOMED, Int., of Danvers, Massachusetts, withsupport fiom NIH ^rant SBIR 2R44 ES03')l8-0i.

Received 17 July 2000; ,icct.-pifd 2(> I'cbruary2001.

Environmental Health Perspectives • VOLUME 1091 NUMBER 81 August 2001 827

Articles • Hu etal.

the use of a K X-ray fluorescence {KXRF}instrument for making in vivo measurementsof bone lead. These measurements are well-validated {12) and have produced data for anumber of previous successful studies onlow-level lead toxicity and hiomarker deter-minants {13-16). We examined the relation-ship of the ALAD polymorphism to boneand blood lead levels. In addition, we con-ducted exploratory analyses to see i f theALAD polymorphism modifies the relation-ship of bone or blood lead levels to any oftheir well-known determinarit.s.

This study was approved by the HumanSubjects C'ommittees of the Brigham andWomen's Hospital and the Harvard Schoolof Public Health.

Materials and MethodsStudy subjects. The NAS is a longitudinalstudy of aging established by the U.S.Veterans Administrat ion in 1963 ( / / ) .Healthy male volunteers from the GreaterBoston (Massachusetts) area were screened atentry and accepted into the study if they hadno history of heart disease, hypertension,diabetes mellitus, cancer, peptic ulcer, gout,recurrent asthma, bronchitis, or sinusitis.Men who presented with either systolicblood pressure >14O mmHg or diastolicblood pressure >'X) mmHg at entry were dis-qualitied. Between 1963 and 1968, 2,280men who met the entry criteria wereenrolled, ranging in age from 21 to 81 years.with tncan age of 42 years at entry. Studyparticipants were asked to return for exami-nations every 3-5 years. At each vLsit, exten-sive physical examinat ion, laboratory,anthropometric, and questionnaire data werecollected. Beginning in 1991, during thecourse of each continuing participant's regu-larly scheduled evaluation ai the Departmentof Veterans Affairs Outpatient clinic inBoston, a fresh blood specimen was obtainedfor measurement of lead, and permission wassought to rake KXRF bone lead measure-ments. Consenting individuals reported tothe outpatient Clinical Research Center ofthe Brigham and Women's Hospital inBoston.

Blood and bone leiui measurements. Weobtained blood samples and analyzed bygraphite furnace atomic absorption spec-troscopy (GF-AAS; F.SA Laboratories.Chelmsford. MA). Values below the mini-mum detection limit of 1 pg/dl, were codedas 0.5 fig/dL. We calibrated the instrumentafter every 21 samples w i th Nat ionalInst i tute o f Standards and Technology(Gaithersburg, MD) materials. We ran 10%of samples in duplicate; at least 10% oftheanalyses were controls and 10% blanks. Intests on reference samples from the Centersfor Disease Control and Prevention (Atlanta,

GA), precision (the coefficienr of variation)ranged from 8% for concentrations from 10to 30 f ig/dl, to 1 % for higher concentra-tions. In comparison with a National Bureauof Standards target of 5,7 pg/dL, 24 mea-surements by this method gave a mean of5.3 fig/dl. (SD I.23Hg/dL).

We took bone lead measurements at twobone sites, the mid- t ib ia l shaft and thepatella, with an AB IOMED KXRF instru-ment (ABIOMED, Inc., Danvers, MA). Thetibia and patella have been targeted for bonelead research because the.se two bones consistmainly of pure cortical and trabecular bone,respectively, whicb represent the two mainbone compartments. A technical descriptionand validity specifications of this instrumenthave been published elsewhere {12,17). Thisinstrutnent provides an unbiased estimate ofbone lead levels (normalized to bone mineralcontent as micrograms of lead per gram ofbone mineral) and an estimate ofthe uncer-tainty associated with each measurement(equivalent to a single SD if multiple mea-surements were taken). Negative estimates ofhone lead concentration may occur for leadvalues close to zero. Tbe technicians whomeasured the bone lead were blinded to theparticipatit's health statu.'i.

ALAD exoN 4 genotypes analysis. Wedetermined the ALAD polymorphism incxon 4 by polymerase chain reaction (I'CR)and restriction fragment length polymor-phism (RFLP), according to the method ofScbwartz et al. {18). We performed !'CRs induplicate with blank controls included ineach .set.

Statistical analyses. We first comparedcharacteristics of subjects who had all data ofinterest, including genotypes and blood andbone lead levels, with subjects who were notincluded in this analysis because of missingdata. Cienc frequencies and tests for Hardy-Weinberg equilibrium were calculated. Only7 (1%) subjects in this population were clas-sified as ALAD 2-2, so we combined ALAD1-2 and ALAD 2-2 into one categor>' {ALAD1-2/2-2) for most of the subsequent analyses.

We compared the distributions of demo-graphic and lifestyle characteristics (as cate-gories) and bone and blood lead levels (ascontinuous variables) by genotype {ALAD I-1 versus ALAD 1-2/2-2)- and tested tbe dif-ferences using chi-square and Student's r-tcststatistics. We adjusted dietar)' vitamin D fortotal caloric intake and divided it into quin-tiles, as we had done before {15). We usedmultivariate linear regression to model deter-minants of tibia lead, patella lead, and bloodlead; we began with core models incltidingdeterminants we had identified in previouslypublished research {13.15}. For the tibia andpatella lead, these core-model determinantsincluded age, educational levels, cumulative

smoking, alcohol consumption, and dietaryintake of vitamin D; for blood lead, thesecore-model determinants included age, cur-rent smoking status, current alcohol con-sumption, dietary intake of vitamin C, andpatella lead [the major internal bone sourceof circulating lead ( / J , /5 ) ] . We repeatedeach of these regressions after adding a termfor genotype {ALAD J-l vs. ALAD 1-2/2-2).

To assess whether genotype may serve asan effect modifier (versus an independentdeterminant) of bone lead, we al.so comparedthe l^-estimates o( core-model determinantsin regressions of tibia and patella bone leadthat were stratified by genotype {ALAD 1-1versus ALAD 1-2/2-2). I f a core-model ^-estimate was markedly different betweengenotypes, we ran an additional exploratoryregression of the entire sample; the regres-sion included a cross-product term thattested for interaction between genotype andthe core-mode! determinant of interest. Thisallowed us to test for the significance oftheinteraction term.

In each ofthe above regressions, we rangeneralized additive models for continuouscovariates to examine the shape of associa-tions with bone lead and blood lead. 'Fhe.scanalyses allowed us to assess for potentialnonlinearities and the need to transformthese covariates.

All data were analyzed using tbe SAS andS-plus statistical packages {I9i. All />-valuesreported are two-sided.

ResultsFhe 776 NAS subjects who participated inthe KXRF bone lead study were comparableto tbe 1,532 NAS subjects who were seen fortheir regularly scheduled visits between 1991and 1995 with respect to distributions ofage. race, education, smt)king status, con-sumption of alcohol, retirement status, andbiood lead level, as noted in an earlier report(IS). O f these 776 NAS subjects, 726 hadinformation on genotype status. We foundno significant differences in distributions ofage, education, alcobol consumption, andblood and bone lead levels among subjectsw i t h and w i thou t genotype (data notshown). Nine of 726 (1%) and 8 of 50 sub-jects (16%) wi th and without genotype,respectively, did not provide information onsmoking status. However, among the sub-jects with information on smoking status,the distribution of never, former, and cur-rent smokers was not significantly differentbetween those with and those witbout geno-type information.

The prevalence (number) af AI^D l-l,ALAD }-2, and ALAD 2-2 was 83,5%(580), 15.5% (108), and 1.0% (7), respec-tively. Tbe distr ibut ion of the differentAIAD genotypes among the 726 NAS study

828 VOLUME 1091 NUMBER 81 August 2001 • Environmental Health Perspectives

Articles • Influence of ALAD on bone and biood lead levels

subjects conformed to Hardy-Wembergexpected frequencies (chi-square = 0.28; df =2;/) = 0.87).

Table 1 shows the demographic charac-teristics and blood and bone lead levels cate-gorized by genotype. The ALAD 1-1genorype was associated with a higher per-centage of current smokers and drinkers than[he ALAD 1-2/2-2 ^enowpe. Blood lead lev-els in this population were relatively low, asexpected, with a mean (SD) of 6.2 (4.1}pg/dL. The mean (SD) blood lead of individ-uals with the ALAD 1-1 genotype [6.3 (4.1)[.ig/dLj, wa.s significanriy higher than that ofthe variant ailele carriers |'i.7 (4.2) [Jg/dL [t-sratisric = 2.11, /> = 0.04)1. "^^ found no dif-ferences in mean bone lead levels, includingtibia and patella lead as well as tibia andpatella lead adjtisted by age. We also did notfind any association berween the variant alleleand the di Tcrcncc between tibia and patellabone lead concentrations (the cortical-trabec-tilar bone lead differential, Table I), a para-meter that had previously been found todiffer by genotype among lead-exposed con-struction workers (^.

As in our previous investigation (/5).older age, lower education level, higher

Table 1. Demographic characteristics and biood andstudy subjects, 1991-1995.

pack-years of cigarette smoking, and lowintake of vitamin D were important predic-tors of bone lead levels in the core regressionmodels (Table 2 Model A, Table 3 ModelA). We found that the ALAD genotype sig-nificantly influenced tibia lead, but notpatella lead. Subjects with the ALAD 1-2/2-2 genotype had tibia lead levels tbat were2.55 pg/g (95% CI, 0.05-5.05) lower thanthose with the ALAD l-l genotype afteradjusting for other covariates (Tahle 2Model B). In the regression models stratifiedby presence or absence of variant allele, hav-ing a less-than-high school education wasassociated with a mean tibia lead level thatwas substantially higher among the ALAD I-1 genotype subjects (8.15 |.ig/g} than amongthe ALAD 7-2/2-2 genotype subjects (4.88|.ig/g; Table 4 Models C and D). In addi-tion, dietary intake of Vitamin D in thehighest versus the lowest quintiles was asso-ciated with a mean difference m tibia leadlevel that was substantially greater among theALAD 1-1 genotype subjects (-7.31 |.ig/g)than among the ALAD 7-2/2-2 genotypesubjects (-3.14 fig/g; Tahle 4 Models C andD). However, no such differences were seenin the models of patella lead stratified by

bone lead concentrations by genotypes among 726

Variable

Age (years)47-5960-69>70

Education^< Higb scboolSome college/tecbnical school> CollegeMissing

Current smoking status^Never smokerFormer smokerCurrent smokerNo information

Cumulative smoking (pack vearsl^01-20>20No information

Currently consuming > 1 alcobolic drinks/dav^YesNo

Blood lead (pg/dL)"

Bone lead (pg/g)''Tibia

Patella

Adjusted tibia"

Adjusted patella''

Patella-tibia difference"

4L4DNMn = 608

100 il6)297149)211 (35)

326 (54)86(141158(28128(41

175(291366 (60159(1018(11

175(291153(25)248(41132(5)

136(22)472 (78)6.3 ±4,1(0, 5, 35)

22.2 ±13.9(-3,19,126)32.2 ±19.9(1.28,165122.3 ±13.1

(-8,5,20,121.3)32.3 ±19.1

(-6.9,28,5,158,7)10 ±12,5

(-0.37, 9, 63]

Genotype) 4MDf-2^-2(n-118)

14(12)61 (52143136)

68 158)15113)31 126)413)

42 (36)72(61)3|3|1(11

42 (36128 (24)44 (37)4(31

15(131103(8715.7 ±4.2(0, 5. 27)

21.2 ±10.9(3,19.5,67130.4 ±17.2(-10,27,85120.5 ±10.6

(0,4,19,4,64,2129.5 ±16,5

(-14,4, 25.7, 79.8)9,2±11.6

(-30,8,41)

p-Vaiue

0,46

0.75

0,04

0,49

0.02

0,04

0.62

0,36

0.17

0.14

0.83

"No, (%). *Mean ± SD (minimum, median, maximum].

genotype (Table 5 Models C and D). Insubsequent models of tibia lead that re-com-hincd the ALAD 1-1 and ALAD 1-2/2-2groups, interaction tetm.s for genotype withless-rhan-high school education and interac-tion terms for genotype with dietary intakeof vitamin D in the highest quiutilc failed toreach statistical significance (data notshown).

In the core model of blood lead, highintakes of Vitamin Cl and iron were associ-ated with low blood lead, as seen before(75). In addition, higher patella hone leadwas as.sociated with higher blood lead andwas the dominant statistical determinant ofblood lead (partial 7^ of 0.14; total adjusted7^ of 0.19). When added to this core model.a term for presence or ahsence of variantallele was not a.ssociated with any meaning-ful change in blood lead. However, in themodels of blood lead that were stratified bythe ALAD genotype (Table 6 Models I.) andR), the effect estimate that was a.ssociatcdwith each microgram per gram increase inpatella bone lead was substantially higheramong the AIAD 1-2/2-2 genotype suhjects(0.15 (ig/dl.) than among \\\*:'ALAD l-lgenotype subjects (0.07 jjg/dL), In a modelthat re-conibined the ALAD I-i and AIAD7-2/2-2genotype subjects (lahlc 7 ModelC), an interaction term for ALAD 1-2/2-2genotype and patella hone lead was associ-ated with a significantly higher blood leadlevel. In the same model, the term for ALAD7-2/2-2genotype alone was associated with asignificantly lower hlood lead level, In asmoothed plot of blood lead in relation topatella lead that was stratified by genotypeand adjusted for other covariates in themodel (Figure 1). this interaction can beappreciated as, among the ALAD 1-2/2-2genotype individuals, a lower blood leadwhen bone lead levels are below 40 pg/g.and a higher blood lead level when bone leadlevels are above 60 ng/g.

DiscussionIn dus suidy, we found no significant dif)cr-ences among participants according to/IL^/) genotype with respect to levels of leadin blood or patella (trabecular) bone.Although a mean blood lead level that wasslightly higher among ALAD I-! thai)among ALAD 1-2/2-2 genotype individuatiwas suggested in the bivariate analyses, thisdifference disappeared in the multivariateregression analysis and may have heencaused by confounding by pattern.^ of alco-hol ingestion and smoking.

On the other hand, we found in the mul-tivariate analyses that tibia (cortical) bonelead levels were signiHcantly lower among theALAD /-2/2-2 than in the AIAD 1-1 geno-type individuals, with a mean difference that

Environmental Health Perspectives • VOLUME 109 I NUMBER 8 I August 2001 829

Articles • Hu et al.

was modest (2.55 ftg/g). If one cotisidersincmbcrship in the lowest edticational class aproxy for significant environmcntal/occLipa-tional lead expo.surc, the differences in the fl-coeHlcient.s associated with this exposurewith respect to tibia bone lead ofthe two/IM/J) genotypes (as seen in the stratifiedregressions) surest that at high levels of leadexposure, AIAD 1-2/2-2 individuals absorbless lead into cortical bone than ALAD l-lindividuals. An alternative explanation isthat ALAD 1-2/2-2 individuals experiencegreater loss of lead from cortical bone thani4I/l£) 7-/individuals.

i-urthermore, we found an interactionbetween patella (trabecular) bone lead andALAD genotype with respect to blood leadlevels. AIJ\D /-2/2-2 genotype individuals

had a steeper trabecular bone lead-bloodlead relationship, with higher blood lead lev-els at a given trabecular bone lead level, butonly when trabecular bone lead levelsexceeded around 60 |-ig/g- Interestingly,there appears to be a crossover phenomenon,with the ALAD 7-2/2-2 genorype individualsshowing significandy lower blood lead levelsat a given trabecular bone lead level whenblood lead levels were < 60 pt^/dL. To ourknowledge, the latter effect has not beenpubiished previously, but there has been lit-tle other research that has examined bothbone and blood lead levels in relation roAIAD genotype, and even less in popula-tions with relatively low levels of exposure.

1 hese particular results are only su^es-tive, because they are based upon a few

Table 2. Predictors of tibia lead ([jg/gl among all subjects in the Normative Aging Study. 1991-iai5 (n

689) ^'JLI

Variable Parameter estimate 95% Cl

(__1H

.__ , Model BParameter estimate 95% Cl

InterceptGenotvpe

[ALAD 1-2/2-2\%. ALAD hnAge lyearslEducations High schoolSome technical schoolMissing

Cumulative smoking (pack-years)1-20>20No information

Alcohola 2 drinks/day

Vitamin D|IU/day|179-262282-375375-589>589

Total model R^

18.55

0.71

7.982,883,43

2.134,750.90

'1.04

-4.71-5.00-3,98-3.44

0,27

0,58-0,84

5 87-10,09-0,16-5.91-1.51-8.36

-0.35^,812,63-6,88

-5.39-3,58

-3.29-1,21

-7,37--2.05-7.89--2.il-6,87--1.10-5,99-0.90

19.01

-2,550,71

7,982.963.28

2,054,59

-1,00

-i.n

-4.79-4.90-3.88-3,550,27

-5.05--0.050.58-0,84

5.87-10,09-0.07-5,99-l,65-a.21

-0.43-4.522,47-6.71

-5.48-3,47

-3.36-1.14

-7.4^-2.13-7,78-2.01-6.75--l,00-6,09--1,01

Table 3. Predictors of patella lead (pg/g) among all subjects in the Normative Aging Study, 199(-,lW3 (" =689)

__,IVlQdelA Model.!Variable

InterceptGenotype[ALAD }-m-2M%. ALAD 1-1]

Age lyearslEducation

s High schoolSome technical schoolMissing

Cumulative smoking (pack-years)1-20>20No information

Alcohol2 2 drinks/day

Vitamin D (lU/day)179-262262-375375-589>589

Total model /?•

Parameter estimate

25,76

0.89

10.233.51

11.72

2,136,632,83

1.93

-7,10-5.49-4.80-5.27019

95% Cl

0,71-1.08

7.17-13.29-0,79-7.804.91-18,53

-1,44-5,693.40-9.85

-3.51-9,16

-1.37-5,23

-10.83--3,27-9.59--l,39-8,90--0,70-9.34--1,19

Parameter estimate

25,94

-1.010.90

10.243.50

11.69

2,106,592.75

1.88

-7.08-5.46-4.75-5,260,19

95% Cl

4.55-2,540.71-1,08

7.18-13.30-0.80-7.804.87-18,50

-1.46-5.673.37-9.82

-3,60-9,09

1.43-5.19

-10,86--3.30-9,56- 1.35• 8.86--0.64-9 33--1 18

ALAD 1-2/2-2 individuals who had veryhigh bone lead levels. Nevertheless, thisobservation suggests that the ALAD poly-morphism modifies movement of lead frombone into blood, with AIAD 1-2/2-2 geim-type individuals pos.sibly being less prone tomobilizing bone lead when bone lead levelsare low. and more prone to mobilizing bonelead when bone lead level.s arc high. Analternative explariaiioii could be that ALAD/-2/2-2 genotype individuals are initiallymore likely to store lead in trabecular bone,but at a certain low saturation point, theyare less likely to store lead in trabccularbone. Under this hypothesis, the interactionat higher levels of lead burden, for example,could be interpreted as reflecting lower bonelead levels at a given blood lead level (withblood lead deriving mostly from exogenoussources) rather than higher blood lead levelsat a given bone lead level (with blood leadderiving mostly from endogenous bone), itis not possible to distinguish these possibili-ties given the cross-sectional nature of thisstudy.

The gene trcquency of die AIAD variantallele in our study was 8%, which is similarto the 6% among 1,278 children reported inNew York (6) and the 8% among 691 mem-bers of a construction trade union reportedin the United States (^. However, it was rel-atively lower than ihe 1 1% reported inKorean lead-exposed workers {« = . 08) {18)and the 13% reported in Germany lead-exposed workers (n = 202) (^. This is likelyattributable to ethnic variation in allele fre-quency.

Two studies have suggested that thev4L4£) genorype may be a selection factor forworking in lead industries. Should this hetrue, it may have implications for the gener-alizability ot findings from occupationaliyexposed groups {9,18). Our research, on theother hand, was conducted in the commu-nity, and the distribution ofthe AIAD poly-morphism conformed closely to theHardy-Weinberg equilibrium. Selection sim-ilar to a healthy worker effect was unlikely inour study {9.IS), and our findings can prob-ably be generalized to low lead-exposed pop-ulations ot men,

In in vitro studies, Battistuxzi et al. (3)examined enzyme activities among difTerentALAD M/»/genotypes and found that meanAIAD cmymc activities (± SD; in l)/fig Hb}are similar among ALAD l-l, 1-2, and 2-2individuals: 52 ± 17 (« = 195), 49 ± 20 {n =43), and 55 ± 7 (n = 5). Subsequently,Bergdahl et al. (.5) found that the presence ofthe A LA L^-2 subunit was associated withmore botind lead when they studied sevenhomozygous wild-type and seven {AIAD)homozygous variant individuals who werelead-exposed workers with blood lead levels

830 VOLUME 1091 NUMBER 8 I August 2001 • tnvironmental Health Perspectives

Articles • Infiuence of ALAD on bone and blood iead leveis

around 30 pg/dL. No significant differencewas found in ALAL') lead-binding among 20unexposed controls whose blood lead leveiswere around 4 [.ig/dL. It i.s possible thac thedifference in AIAD-hound lead in erythro-cytes may be detectable only at higher leadlevels. In this case, the ALAD-2 protein mayeither bind lead more tightly than theALyiD'l protein or have more bindmg sites.Alternatively, the lack of difference may beexplained simply by the difficulty inherent inquantitating binding at low lead levels (5).

Zienisen et al. {7) examined blood leadlevels in different ALAD genotypes among202 lead-exposed workers whose mean (±SD) (jig/dL) blood lead levels were 40 (± 17){7). They found chat blood lead levels (mean± SD, pg/dL) among the ALAD 1-1 {n =160), ALAD 1-2 {n= il), ALAD 2-2 {n =10) groups were 38 ± 17, AA ± 17, and 56 ±18, respectively, although these were not sig-nificantly different. Wetmur et al. (6) exam-ined the association of ALAD genotype andblood lead levels among 202 lead workers inGermany and 1,278 children in New York.They found that the blood lead levels among/4/,y^D-2 carriers had median values chatwere about 9 to II (.ig/dl. greater than simi-larly exposed individuals who were homozy-gous for the ALAD-l allele. These fmdingssuggested that che ALAD-2 polypeptidebinds lead more cightly and effectively thanALAD-l. These studies had mean blood lev-els that were higher than 20 and 40 pg/dLamong environmentally exposed chddrenand lead-exposed workers, respectively.Smith et al. {8) investigated the associationbetween the presence of ALAD-2 a\\t:\e andlead concentrations in blood and boneamong 688 members of a construction tradeunion. 'I'hcy did not find a significant differ-ence in mean blood lead concentrationsbetween ALAD-2 carricTs (n = 96; mean ±SD. 7.7 ± 3.5 pg/dL) and chose homozygousfor the ALAD-l allele {?7 = 592; mean ± SD,7.8 ± 3-6 (ig/dL). However, they found aborderline statistically significant difference(/» = 0.06) by subtracting the tibia lead con-centrations from the patella lead concentra-tions for each subject between these twogroups (/1/./4D-2 carriers: ri = 21; mean ±SD, 8.6 ± 9.5 ^ig/g; homozygous ALAD-lallele: « = 101; mean ± SD, 3.4 ± 12.0 pg/g)among a subset of 122 study subjects. Bloodlead levels in the union members were rela-tively low (mean ± SD, 7.8 ± 3.6 pg/dL).and die authors suggcsced chat ALAD poly-morphism may modify lead kinetics butonly at higher blood lead levels, whichwould be consistent with che in vitro studybyRergdahlecal. (5).

Recently, two studies investigated themodulating effect of AIAD polymorphismon both blood and bone lead in lead smelter

Table 4. Predictors of tibia lead (pg/g) among participants in the Normative Agingfied by genotype.

idy; 1991-1995, strati-

Variable

nterceptAge (years)education< High schoolSome technical schoolMissing

Cumulative smoking (pack-years)1-20>20No information

Alcohol> 2 drinks/day

Vitamin D (lU/day)179-262262-37537&-589>589

Total model f?

Table 5, Predictors of patellastratified by genotype.

Variable

InterceptAge (years)Education< High schoolSome technical schoolMissing

Cumulative smoking (pack-years)1-20>20No information

Alcohol> 2 drinks/day

Vitamin D (lU/day)179-262262-375375-589>589

Total model fl'

Table 6. Predictors of bloodstratified by genotype.

Variable

InterceptPatella lead Ipg/g)Genotype{ALAD hUs. ALAD h2/2-2\

Interaction

Model CALAD 1-Un =

Parameter estimate

18.640.75

8.152.643.19

1.904.70

-0.12

-0,97

-4.58-4.5513.92-3.140.Z8

580)95% Cl

0.60-^.89

5.79-10.600.78-6.072.10-8.49

-0.90-4.692.31-7.08

-5.12^.88

-3,44-1,50

-7.49--1.66-7.81--1.28-7.16--0.68-5.97--0.30

lead (pg/g) among participants in the

Model CALAD 1-2 [n =

Parameter estimate

26 010.91

9,692.98

11.12

2.427,364.72

1.84

-7.26-5.11-6.40-4.65019

580)95% Cl

0.71-1.12

5,79-10.50-1.81-7.77-2.10-8,49

-1.62-6.463.71-11.02

-2.24-11.67

1.79-5.47

-n,40--3.i2--9.74-- 0.47

-11.30-'! .76-9,20- n 11

ead (pg/dL) among participants in the

Model DALAD 1-1 in=

Parameter estimate

5.120.07

580)95% Cl

0,05-0.09

Also in model: age, smoking, alcohol ingestion, vitamin C intake, iron intake.

Model DALAD 1-2/2-2 {ti

Parameter estimate

19.510.44

4.883.693.61

3.793.92

-4.14

-1,63

^4.91-6.50-3.26-7.310.22

/ // T--Mormatiye Agifig/Studv

Mode!DALAD \-2/2-2\n

Parameter estimate

23.220.83

12.445.86

14,88

2.674.34

-8.07

2.35

"^.76-7.021.94

-6.370.27

\l 0 rmative^^W/lgStud V

rfrlModel E

AiAU}'2/2-2 [Parameter estimate

3.480.15

= 109)95% Cl

017-0.72

0.17-9.582.58-9.95

11.37-18.59

-1.42-9.000,58-8.42

14,27-5.99

-7,08-3.82

11.4&-1.6712.33--0.68-9.23-2.7113.26---1.36

1991-1995,

= 109)95% Cl

0.42-1.23

5.58-19.30-3,79-15.50-3,50-33.27

'4.96-10,30-2.53-11.21^4.85-8.70

-6.05-10.75

15.47-3.9515.75-1.71^6.93-10.8215.71-2.98

1991-1995,

1= 109)95% Cl

0.10-0.19

Table 7. Predictors of blood lead (pg/dL) among all subjects in the Normative Aging Study, 1991-1995 (n =689).

Model AParameter

Variable

InterceptPatella lead (pg/g)Genotype[ALAD 1-1 vs. ALAD h2/2-2\Interaction

estimate 95% Cl

4.900.08 0.07-0.10

Model B Model CParameterestimate

4.9S0.08

-0 27

Parameter95% Cl estimate

5.190.07-0.10 0.07

-1 05 0.5n 2240.06

95% Cl

0.06-0.09

-3.83--0.650.02-0.11

Also in model: age, smoking, alcohol ingestion, vitamin C intake, iron intake.

Environmental Health Perspectives • VOLUME 1091 NUMBER 8 I August 2001 831

Articles • Hu et al.

/ ALAOl-l

i-i ALADI-2/2-2

Patella lead |iig/g)

Figure 1. Smoothed line of blood lead versuspatella lead by genotypes In 689 community-exposed men, with adjustment for age, alcoholconsumption, current smoking, vitamin C intake,and iron intake: the Normative Aging Study,1991-1995.

workers (9J0). Tht average blood lead levelsin both groups of lead smelter u-orkers wereapproxiniLitely 20-30 |.ig/dl., wW\c\\ is higherthan in our .study (mean. 6.2 |ag/dl.) (Tables2 and 4). Bergdahl ct al. (9) did not find anysignificant association between ALAD geno-type and blood or bone lead levels among 89lead-exposed workers. They also examined34 unexposcd Vi'orkers whose median bloodlead level wa.s 4 fig/dL and found no differ-ence in blood lead levels between 24 ALADI-} and 10 ALAD /-2 individuals. Becauseihe sample size was small in this study, it isdifficult to interpret these findings.However, the authors did find that the con-centrations of urinary calcium and the rarioof urinary creatinine/scrum creatinine weresignificandy lower in 7 ALAD 1-212-2 thantho.se of 82 ALAD l-l lead workers [medi-ans: urinar\' calcium (miliigrams per liter) 76vs. IHS; rhe ratio urinary creatinine/serumcreatinine 84 vs. 180]. This study suggestedthe presence of yl/.>)D allele-specific differ-ences in kidney function.

In another study, Fleming et al. {10)found the average blood lead levels were22.9 ± 0.4 and 25.2 ± 1.0 pg/dl. in 303ALAD l-l and 65 ALAD 1-2/2-2 icme leadworkers {p < 0.05). But they did not findany significanr difference in tibia bone leadby genotype (mean ± SD, 41.2 ± 1.8 inALAD l-'l and 42.7 ± 3.4 in ALAD 1-2/2-2). In addition, chey did not find any appar-ent difference in the contribution of bonelead (either tibia or calcaneus lead) to bloodlead levels by genotype. However, using actimulativc blood lead index (CBl.l) for each

worker, on the basis of individual blood leadhistories and bone lead measurements toestimate total lead body burden, they foundin linear regressions that the slopes of bonelead ro CBLi were steeper among ALAD l-lwotkers. The more efficient uptake of leadfrom blood into the bone of workers withthe ALAD l-l genotype is consistent wirhour findings and suggests a decreased trans-fer of blood lead into bone in ALAD 1-2/2-2lead workers {lO).

In our study, we found that patella leadis the major predictor of blood lead in thisaging community-exposed population andchat the ALAD polymorphism significantlymodifies this association. When patella leadwas > 60 pg/g, blood lead levels in ALAD 2carriers were higher than those in ALAD l-lindividttals. However, when patella lead was< 40 |.ig/g, blood lead ievels in ALAD l-lindividuals were higher than chat in AlAD 2carriers. Otir results imply that when bioodlead levels are relatively low (< about 8jig/dL), ALAD l-l individuals wiM havehigher blood iead levels than AlAD 2 carri-ers. This finding suggests that the modulat-ing effect of the ALAD polymorphism onblood lead depends on the bone lead bur-den. Again, the finding is tentative and mustbe verified in other community-exposedpopulation studies.

We conclude that the ALAD polymor-phism may modify the exchange of leadbetween blood and bone. This, in turn, maymodify an individual's ultimate risk for toxic-ity. Indeed, several studies have found thatthis ALAD polymorphism modifies indica-tors of possible lead toxicity, such as kidneyfunction (8,9) and reproductive {20) andneuropsychologic function {21). The neteffect on clinical function of the ALAD-2allele may be detrimental [e.g., on renal func-tion, as seen by Bergdaht et at. (9i and Smithet al. { 1 or protective [e.g., on neurop.sycho-logic function, as seen by Bellinger et al. (21)and on sperm count as seen by Alexander etal. (20)], possibly depending on the impactof this polymorphism on the tissue distribu-tion of lead. Further research is needed todefine precisely the mechani.sm of (unctionand the potential impact of the ALAD poly-morphism on lead kinetics and toxicity.

REFERENCES AND NOTES

1. Wetmur JG, Kava AH. Plawinska M, Desnick RJ.Molecular characterization of the human fi-aminolewulinicacid dehydratase. 2 \ALAD2\ allele: implications foi molec-ular screening of individuals for geneirc susceptibility tolead poisoning. Am J Hum Genei 49:757-763 (19S11.

2. Onalaja AO, Claudio L Genetic susceptibility to lead poi-soning. Enwiron Health Perspect 108(suppl l):23-38(2XI0).

3. Battistuzzi G, Petrucci R, Silwagni L tJrbani FR, Caiola S,6-Aminolevulinic acid dehydratase: a new genetic poly-morphism in men, Ann Hum Genet 45:223 229(19811.

4. Wetmur JG, Bishop DF, Canielmo C. Desnick RJ Human6-aminolevulinic acid dehydratase. nucieotide sequenceof a fjll-length cDNA clone. Proc Nail Acad Sci USA83:7703-7707119861.

5. Bergdahl lA, Gnibb A, Schutz A, Oesnick RJ, Wetmur JG,Sassa S, Skerfving S Lead binding to ^-aminol8Wulinicacid dehydratase {ALADi in human erythrocytes.Pharmacol Toxicol 81:153-15811997).

6. Wetmur JG. Lefinert G, Desnick RJ. The I'l-HmmolevulinicBcid dehydratase polymorphism: higher blood lead levelsin lead workers and enwtronmentally exposed childrenwith the )-2and 2-2isozymos. Environ Res 56:109-119(1991).

7. Ziemsen B, Angerer J, Lehnert G. Benkmann HG, GoeddeHW. Polymorphism ol h aminolevulinic acid dehydratasein lead exposed workers. Int Arch Occup Environ Health58:245-247(19861.

8. Smith CM, Wang X. Hu H, Kelsey KT A polymofphism inthe iVaminolevulinic acid dehydratase gene may modifythe pharmacokinetics and toxicity of lead. Environ HealthPerspect 103:248-253 (19951.

9. Bergdahl lA, Gerhardsson L. Schut; A, Wotmur JG,Skerfving S. Delta-aminolevulinic acid dehyd'atase poly-morphism: influence on lead levels and kidney functionin humans. Arch Environ Health 52:91-96 (1997).

10. Fleming DEB, Chettle DR, Weimuf JG, Desnick RJ, RobinJP, Bciilay D. Richard NS, Gordon CL, Webber CE. Effectof the d-aminolewulinate dehydratase jjolymorphism onthe accumulation of lead in bone and blood in leadsmelter workers. Environ ftes 77:49-61 1998.

11. Bell B, ROSE CL, Damon A, The Normative Aging Study,an interdisciplinary and longitudinal study ol health andaging. Aging Hum Dev 3:4-17 (19721,

12. Hu H, Bone lead as a new biologic marker of lead dose:recent findings and implications (or public health.Environ Health Perspect 106(suppl 41:961 967 (1998),

13. Hu H, Payton M, Korcick S, Aro A, Sparrow D, Weiss ST,Rotnit^ky A Determinants o( bone and blood lead iHvelsamong community-exposed middle-aged to elderly men:the Normative Aging Study, Am J Epidemiol 144:749-759(1996),

M, Hu H. Aro A. Payton M, Korrick S. Sparrow D, Weiss ST,Rotnizky A. The relationship of blood and bone lead tohypertension among middle-aged to elderly men, J AmMed Assoc 275:1171 1176(1996).

15, Cheng V, Willett W, Schwam J, Sparrow D, Weiss ST.Hu H, The relationship of nutrition lo bone and hlood lead(evels in middle-aged to elderly men, the NormativeAging Study. Am J Epidemiol 147.1162-1174 (ISSB).

16, Cheng V, Sctiwaru J. Vokonas P, Weiss ST, Aro A, Hu H.Electrocardiographic conduction disturbances m associ-ation with low level lead exposure: the Normative AgingStudy. Am J Card.ol 82:594-599 (1998).

17, Burger D, Morsil lo P, Adams B. Hu H, Milder FL,Automated instrument for making K-X-ray fluorescencemeasurements in human bone. Basic Life Sci 55:287 293(1990).

18, Schwartz BS, Lee BK, Stewart W. Ahn KD, Springer K,Kelsey K, Associations of iS-aminolevulinic acid dehy-dralase genotype with plant, exposure duration, andblood lead and zinc protoporphyrin levels in Korean leadworkers. Am J Epidemiol 542:738 745 (1995),

19, SAS Institute Inc. SAS Language Guide (or PersonalComputers, Release 6,03 Edition. Gary, NC:SAS InstituteInc, 1988,

20, Alexander BH, Checkoway H, Costa-Mallen P, FaustmanEM, Woods JS, Kelsev KT, van Netten C, Costa LG.Interaction of blood lead and h-aminolevuiinic acid dehy-dratase genotype on markers of heme synthesis andsperm production tn lead smelter workers. EnvironHealth Perspect 106:213-216 (19881

21 Bellinger D, Hu H. Titlebaum L, Needleman HL.Attentional correlates of dentin and bone lead levels inadolescents. Arch Environ Health 49:98-105 1994

832 VOLUME 1091 NUMBER 81 August 2001 • Environmental Health Perspectives