1 Department of Neurology, Kennedy Krieger Research Institute, Johns Hopkins Medical Institutions, Baltimore, MD.
2 Department of Epidemiology, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, MD.
3 Division of Occupational and Environmental Health, Department of Environmental Health Sciences, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, MD.
4 Department of Medicine, School of Medicine, Johns Hopkins University, Baltimore, MD.
5 Division of Toxicological Sciences, Department of Environmental Health Sciences, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, MD.
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ABSTRACT |
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blood pressure; genetic predisposition to disease; genetics; lead; Na(+)-K(+)-exchanging ATPase
Abbreviations:
ATP1A2, gene for the 2 subunit of sodium-potassium adenosine triphosphatase; Na+-K+ ATPase, sodium-potassium adenosine triphosphatase; RFLP, restriction fragment length polymorphism; SD, standard deviation
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INTRODUCTION |
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Several epidemiologic studies have suggested a relation between exposure to lead, particularly at blood concentrations less than 40 µg/dl, and small increases in blood pressure, but other studies have failed to find a relation (4, 5
). The conflicting results among studies may be attributed to demographic differences between the populations studied, variation in genetic susceptibility, and limitations of the measures used to define lead exposure.
The mechanism of action for the effect of lead on blood pressure has not been established. However, inorganic lead is a potent inhibitor of sodium-potassium adenosine triphosphatase (Na+-K+ ATPase) in vitro. Lead inhibits Na+-K+ATPase in mammalian tissues (610
). Moreover, early epidemiologic studies observed lower enzyme activity in eryth-rocyte membranes of workers with elevated blood lead levels (11
13
). Although data are not conclusive, lower levels of Na+-K+ ATPase activity in erythrocytes and leukocytes have been reported in hypertensive individuals (14
17
), and an inverse association between Na+-K+ATPase activity and blood pressure has been reported (18
).
Na+-K+ ATPase is an integral membrane protein that catalyzes the active transport of Na+ and K+ across the cell membrane. The enzyme is composed of a catalytic subunit and an associated ß subunit that is necessary for proper enzyme function. There are three distinct isozymes for the
subunit that have variable distributions in different tissues (19
). The
1 subunit is ubiquitous and predominates in the kidney; the
2 subunit is most prevalent in the brain, heart, and muscle cells (19
). Restriction fragment length polymorphisms (RFLPs) in the Na+-K+ ATPase genes exist (20
25
). The RFLPs are of interest as markers of susceptibility to hypertension and as modifiers of the influence of lead on blood pressure. Indeed, studies of the normotensive offspring of hypertensive parents indicate a possible role for Na+-K+ ATPase (26
29
).
Differences in the prevalence of genetic polymorphisms for Na+-K+ ATPase genes may explain variation in results of studies examining the effect of lead on blood pressure. We investigated the association of two RFLPs in the gene for the 2 subunit of Na+-K+ ATPase (ATP1A2) with blood pressure and hypertension, as well as possible effect modification by genotype on the lead-blood pressure relation.
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MATERIALS AND METHODS |
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Selection and recruitment of study subjects
The study was approved by the Committee on Human Research of the Johns Hopkins School of Hygiene and Public Health, and subjects provided written informed consent. The prospective study focused on individuals employed in the organolead manufacturing area (termed the tetraethyl lead facility), which was involved in the manufacture of tetraethyl lead from 1923 to 1991 and the manufacture of tetramethyl lead from 1960 to 1983 (30). Workers were eligible for recruitment if they had been employed at the facility on or after January 1, 1950, were male, were between 40 and 70 years of age in 1995, were not too ill to continue participation, resided in the state of New Jersey, and had a tibia lead measurement. For the current study, a sample of 272 subjects was selected from 543 eligible workers. To ensure sufficient numbers of individuals with high exposure levels, half of the sample consisted of participants with the longest duration of exposure to lead (10.144.3 years), and the remainder were randomly selected from the balance of enrolled participants.
Data collection was completed for 226 (83 percent) of 272 former lead workers who were selected for this study. Individuals were asked to participate in the blood pressure study at a supplemental visit during which tibia bone lead was measured. Twenty-five individuals refused to participate or our attempts to obtain a blood specimen were unsuccessful, and 21 individuals were not invited to participate because blood specimens could not be processed. The final study sample was comparable to the enrolled, eligible cohort with respect to data collected at the initial visit, including age, systolic and diastolic blood pressure, and blood lead concentration (data not shown).
Data collection
Data on demographic and personal characteristics and lead exposure were obtained during the initial visit of the prospective study. A blood sample for determination of blood lead level was also drawn. At the supplemental visit during the third year of the study, tibia lead, blood pressure, and weight were measured, and a 10-ml venous blood sample was obtained. Buffy coat was isolated, frozen in liquid nitrogen vapor on-site, and transported to the Johns Hopkins School of Hygiene and Public Health for storage at -70°C.
Exposure assessment and dose measurements
The methods used for assessing exposure to organic and inorganic lead (30) and validating exposure estimates (31
) are described in detail elsewhere. Interview and personal industrial hygiene data were combined to derive individual measures of cumulative lead exposure (µg/m x years), duration of lead exposure (years), and lifetime average exposure (i.e., cumulative lead exposure/exposure duration, µg/m3).
Methods for assessment of lead dose are described in detail elsewhere (32). Blood lead levels were measured at a commercial laboratory that was certified by the Occupational Safety and Health Administration, using the National Institute for Occupational Safety and Health's standard addition method (33
), by graphite furnace atomic absorption spectrophotometry. Tibia lead was estimated by a 30-minute measurement using 109Cd K x-ray fluorescence at the mid-tibial shaft (34
37
).
Blood pressure measurement
Blood pressure (systolic and fifth Korotkoff phase dia-stolic) was measured using a Hawksley random-zero sphygmomanometer and a protocol compatible with the recommendations of the American Society of Hypertension (38). Three measurements using an appropriately sized cuff were taken 5 minutes apart with the subject sitting. The mean of the three readings was used in the analysis. Blood pressure measurements for all subjects were taken by one trained technician.
Na+-K+ ATPase RFLP genotyping
RFLP genotypes for the Na+-K+ ATPase 2 subunit were determined using Southern blot techniques adapted from Deriaz et al. (39
). DNA (10 µg), extracted from thawed buffy coat specimens, was digested at 37°C with Bgl II restriction enzyme, precipitated, separated electrophoretically (0.8 percent agarose), and transferred overnight to nylon membranes. The blots were prehybridized for 4 hours at 68°C in a buffer containing 250 mm Na2HPO4, 1 mm ethylenediaminetetraacetic acid, 7 percent sodium dodecyl sulfate, and 0.1 mg/ml salmon sperm DNA. Membranes were hybridized sequentially with genomic DNA probes specific for the
2 gene provided by Dr. J. B. Lingrel (University of Cincinnati, Cincinnati, Ohio). One probe, a 2.5-kilobase EcoR1 fragment of the 5' end of the
2 gene, includes the first exon. The other probe, a 1-kilobase EcoR1 fragment at the 3' end, includes exons 21 and 22. After overnight hybridization in the same buffer at 68°C with 32P-labeled probe (0.5 µCi/ng probe), the membranes were washed four times (10 minutes at 25°C, 60 minutes at 25°C, 60 minutes at 50°C, and 60 minutes at 68°C) in a buffer containing 19.5 mm NaCl, 1 mm NaH2PO4, 0.1 mm ethylenediaminetetraacetic acid, and 0.1 percent sodium dodecyl sulfate (pH 7.7). Autoradiograms were developed after 510 days' exposure at 70°C. Three genotypes for each probe were identified based on the lengths (kilobases) of the DNA fragments, estimated using a HindIII/EcoR1 DNA digest. The genotypes were 4.3/4.3, 4.3/10.5, and 10.5/10.5 for the 1.0-kilobase ATP1A2 (3') probe and 8.0/8.0, 8.0/3.3, and 3.3/3.3 for the 2.5-kilobase ATP1A2 (5') probe. Genotypes for 20 percent of the individuals were assayed twice, and the genotype results were found to be 100 percent reproducible.
Statistical analysis
Linear regression was used to evaluate the relations of systolic and diastolic blood pressure with lead dose. Covariates were age (years), race (African-American, non-African-American), body mass index (weight (kg)/height (m)2), use of antihypertensive medications (any, none), a diagnosis of diabetes, alcohol consumption (lifetime reported number of drinks, in quartiles), smoking (nonsmoker, current smoker, or past smoker), and season of blood pressure measurement (January/February/March, April/May/June, July/August/September). Dummy variables for genotype at each RFLP site (ATP1A2 (3') and ATP1A2 (5')) and an interaction term for genotype lead dose were included. Each RFLP (ATP1A2 (3') and ATP1A2 (5')) was examined separately.
The range of lead dose was smaller among subjects who were homozygous for the 10.5-kilobase allele at the 3' RFLP site. Therefore, the analysis of effect modification by genotype was repeated after we limited the analysis to subjects with lead levels present in all genotypes. This resulted in the exclusion of 12 individuals. Since antihypertensive medications alter blood pressure, the analysis was repeated after the exclusion of 56 subjects who reported taking antihypertensive medication.
Relations between hypertensive status, RFLP genotype, and lead dose were examined by means of logistic regression. The analysis used two definitions for hypertension. First, we identified 66 hypertensive subjects by defining hypertension as systolic blood pressure >160 mmHg, diastolic blood pressure >95 mmHg, or an affirmative response at the first visit regarding current use of antihypertensive medications. Second, we identified 81 individuals by defining hypertension as systolic blood pressure >150 mmHg, diastolic blood pressure >89 mmHg, or the use of medications to control blood pressure. The logistic regression analysis modeled 2 genotype using dummy variables for each
2 genotype, and as an ordinal predictor variable (zero, one, or two copies of the less common allele).
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RESULTS |
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The ATP1A2 (3') RFLP significantly modified the association between blood lead and systolic blood pressure (p = 0.01) (table 3). The slope for the relation between systolic blood pressure and blood lead was significantly greater for the 10.5/10.5 genotype compared with the other two groups. The regression coefficients for genotypes 4.3/4.3 and 4.3/10.5 did not significantly differ from each other, and results are presented for the two genotypes combined. Systolic blood pressure increased by 0.5 mmHg and 6.1 mmHg with each 1-µg/dl increase in blood lead level for genotypes 4.3/4.3 and 4.3/10.5 combined and genotype 10.5/10.5, respectively (figure 1). Exclusion of subjects with blood lead values above those in the 10.5/10.5 group or of subjects taking antihypertensive medication did not alter the relations. Twenty-six of the 56 subjects taking antihypertensive medication were heterozygous and three were homozygous for the 10.5-kilobase uncommon allele, indicating that the lead-genotype interaction is not solely due to the association between genotype and hypertension.
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Hypertension and ATP1A2 (3') genotype
Sixty-six (30 percent) of the 220 workers met the definition of hypertension. Mean age and education for persons with hypertension were comparable to those for normotensive individuals. Hypertensive participants had a significantly higher body mass index (31.1 (SD 5.1) vs. 28.7 (SD 4.3); p = 0.0003), were more likely to be former users of tobacco (56.1 percent vs. 51.3 percent; p > 0.05) or alcohol (28.8 percent vs. 23.4 percent; p > 0.05)), and were more likely to be African-American (9.1 percent vs. 4.6 percent; p > 0.05). There were no significant differences in mean blood lead level by hypertensive status. The prevalence of hypertension was significantly higher among workers with the 10.5/10.5 genotype (p = 0.03), and a larger proportion of these subjects were using antihypertensive medication (table 1).
The 10.5/10.5 genotype was directly associated with hypertension (adjusted odds ratio = 7.7; 95 percent confidence interval: 1.9, 31.4) (table 4), after adjustment for age, body mass index, and estimated lifetime alcohol consumption. The association of ATP1A2 (3') genotype with hypertension was not sensitive to either the method of modeling genotype or the definition of hypertension. For example, when ATP1A2 (3') genotype was modeled as an ordinal variable (i.e., zero, one, or two 10.5-kilobase alleles), genotype was significantly associated with hypertension (adjusted odds ratio = 1.9; 95 percent confidence interval: 1.1, 3.3). Moreover, the 10.5/10.5 genotype remained a significant risk factor when a broader definition of hypertension was used (see "Materials and Methods") (adjusted odds ratio = 5.1; 95 percent confidence interval: 1.3, 20.6). Blood or tibia lead levels were not associated with hypertension when added to the model, and there was no effect modification by ATP1A2 (3') genotype on the relation between blood or tibia lead and hypertension.
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DISCUSSION |
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The prevalence of the 10.5-kilobase allele at the 3' RFLP site in this sample was higher than that reported in a Canadian study (25 percent vs. 18 percent), while the prevalence of the 3.3-kilobase allele at the 5' RFLP site was lower (9 percent vs. 12 percent) (39). However, the distri-bution of genotypes at either locus did not vary from the Hardy-Weinberg equilibrium. Cell frequencies in a 2 x 2 table of individuals who were homozygous or heterozygous for the more common allele at each of the two RFLP sites (upper left of table 2) suggested that the two loci on the ATP1A2 gene may be linked, albeit not tightly. Indeed, while hypertension was associated with genotype at the 3' RFLP site, no health endpoints were related to genotype at the 5' RFLP site. In contrast, Rankinen et al. (40
) found significant linkage disequilibrium between the two sites in a sibling-pair analysis of participants in the Quebec Family Study. The 5' genotype was not directly related to blood pressure in the Canadian study, although it was associated with other hemodynamic phenotypes. The 3' genotype was related to resting systolic blood pressure, 12-year change in systolic blood pressure, and exercise diastolic blood pressure, and the 3'/5' haplotype was related to diastolic blood pressure. The findings of our study and of the Canadian study are in conflict with those of Shull et al. (41
), who failed to observe segregation of polymorphisms in the
2 and ß genes with essential hypertension, a study that only involved three families and no individuals homozygous for the uncommon allele.
It is possible that the relations between genotype and hypertension and genotype and race may be due to differential survival in this lead-exposed occupational group. However, while study subjects had past occupational exposure to lead, they had not been exposed to lead occupationally for an average of 14 years. Indeed, the range of blood lead in study participants was similar to that reported for other middle-aged or elderly males residing in the eastern United States with environmental exposure to lead (42). The study sample was selected from all previously lead-exposed workers, minimizing selection bias. Therefore, genetic variation in the
2 subunit of Na+-K+ ATPase may contribute significantly to the prevalence of hypertension in the general population, but this conclusion must await studies of ATP1A2 (3') genotype and hypertension in subjects without past occupational lead exposure. The association between genotype and race probably did not result from selective loss. African Americans with the 10.5/10.5 genotype would have been more strongly subject to selection given the association of race and genotype with hypertension, a risk factor for cardiovascular disease and mortality (b43
). Therefore, we do not think that differential survival explains the higher prevalence of the 10.5 allele among African Americans compared with Caucasians.
The difference in allele frequency by race raises the possibility that the 2 subunit of Na+-K+ ATPase plays a significant role in the pathogenesis of hypertension among African Americans. A higher proportion of African Americans are hypertensive compared with other racial groups (44
). Most of the hypertension in African Americans is salt-sensitive and associated with low plasma renin, conditions that appear to be under genetic control (44
). Moreover, mean blood lead levels of African Americans are higher than those of Caucasians (45
). Consequently, African Americans may be especially affected by the interaction between lead and ATP1A2 (3') genotype and elevations in systolic blood pressure. Unfortunately, the number of African Americans or individuals of other racial groups in the sample was too small to examine the relations between genotype, lead, and blood pressure by race.
Little is known about the number, identity, and function of genes involved in blood pressure control. Two rare autosomal dominant forms of hypertension have been described, glucocorticoid-suppressible hyperaldosteronism and an abnormality of sodium reabsorption in the renal distal tubule (Liddle's syndrome) (3). Several genes that may increase susceptibility to hypertension have been studied, including the renin, angiotensin I converting enzyme, angiotensin type II receptor, angiotensinogen, and adducin genes. While none have been definitively ruled out, strong evidence of linkage with hypertension has been documented for the angio-tensinogen and adducin genes (46
49
). Angiotensinogen is the first substrate in a regulatory cascade resulting in the release of aldosterone from the adrenal cortex, a hormone that regulates sodium retention and excretion (50
). The polymorphism in the adducin gene was associated with a measure of salt sensitivity in 86 hypertensive patients (48
). Na+-K+ATPase is another example of a gene that regulates homeo-stasis and may influence blood pressure.
The effect, if any, of the nucleotide change identified by the Bgl II endonuclease at the 3' RFLP site on enzyme function is not clear. The change has not been specified, and its location (exon or intron) is not known. The polymorphic site is identified in Southern blots by a 1-kilobase probe containing exons 21 and 22. Exons 1922 code for hydrophobic domains in the protein that may be transmembrane domains (22). Site-directed mutagenesis studies indicate that changes in the transmembrane region can alter enzyme kinetics (51
54
).
The role of Na+-K+ ATPase in the mechanism of blood pressure elevation is not known. In experimental studies, inhibition of Na+-K+ ATPase results in increased peripheral vascular resistance and enhanced vascular responsiveness to vasoconstrictor hormones (55, 56
). Na+-K+ ATPase inhibition in the brain also may influence blood pressure. A compound that is structurally similar to the Na+-K+ ATPase inhibitor, ouabain, has been identified in the human circulation and may originate in adrenocorticoid tissue (57
). Circulating levels of "endogenous ouabain" are increased in many forms of hypertension, and its infusion into rats elevates blood pressure (57
). Increased levels of endogenous ouabain in serum and in the hypothalamus could inhibit Na+-K+ ATPase, increase sympathetic activity and aldosterone levels, and elevate blood pressure. The association between hypertension and an RFLP in the gene for the
2 subunit, which is present in significant quantity in the brain, is consistent with this hypothesis. Future research is needed to identify intermediate phenotypes that vary with
2 genotype.
The results of this study provide additional evidence that lead dose is associated with elevated blood pressure. The findings suggest that Na+-K+ ATPase genotype can alter this relation. Na+-K+ ATPase may also have a direct role in the pathophysiology of hypertension. However, because of the small number of individuals with the susceptible genotype, these observations must be considered preliminary.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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