eNOS genotype-dependent correlation between whole blood lead and plasma nitric oxide products concentrations

Nitric Oxide 14 (2006) 58–64 www.elsevier.com/locate/yniox

eNOS genotype-dependent correlation between whole blood lead and plasma nitric oxide products concentrations Fernando Barbosa Jr. a, Valeria C. Sandrim a, Juliana A. Uzuelli a, Raquel F. Gerlach b, Jose E. Tanus-Santos a,¤ a

Department of Pharmacology, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Av. Bandeirantes, 3900, 14049-900, Ribeirao Preto, SP, Brazil b Department of Morphology, Estomatology and Physiology, Dental School of Ribeirao Preto, University of Sao Paulo, Av. do Cafe, S/N, 14040-904, Ribeirao Preto, SP, Brazil Received 20 September 2005 Available online 2 November 2005

Abstract Experimental data indicate that lead exposure decreases nitric oxide (NO) availability. However, no previous study has examined whether lead exposure aVects plasma nitrite/nitrate (NOx) concentrations in humans. In addition, the T¡786C polymorphism aVects endothelial NO synthase (eNOS) expression and endogenous NO release. Here, we investigated whether there is an association between the circulating concentrations of NOx and the concentrations of lead in whole blood (B-Pb) and in plasma (P-Pb) from lead-exposed subjects. In addition, we also evaluated whether eNOS genotype for the T¡786C polymorphism aVects NOx concentrations in lead-exposed subjects. We studied 104 subjects exposed to lead who were non-smokers, 18–60 years of age, and not alcohol consumers. Genomic DNA was isolated from blood samples and genotypes for the T¡786C polymorphism were determined by PCR and restriction fragment length digestion. Circulating NOx was determined by chemiluminescence. B-Pb and P-Pb were determined by inductively coupled plasma mass spectrometry and by graphite furnace atomic absorption spectrometry, respectively. No signiWcant correlations were found between NOx and B-Pb and P-Pb measured in the 104 subjects (all P > 0.05). However, while no signiWcant correlation was found in subjects with TT genotype, a negative correlation was found between plasma NOx and B-Pb (r D 0.230, P D 0.048) and P-Pb (r D 0.194, P D 0.110) in subjects from TC+CC genotypes group. Our study shows a negative correlation between plasma NOx concentrations and B-Pb in carriers of the “C” allele, thus suggesting a possible mechanism possibly involved in lead exposure-induced increase in the susceptibility to cardiovascular diseases.  2005 Elsevier Inc. All rights reserved. Keywords: Endothelial nitric oxide synthase; Genotype; Lead; Nitric oxide; Polymorphisms

Lead exposure is widely recognized as a serious environmental health problem. In this regard, a causal association between lead exposure and increased cardiovascular risk has been strongly suggested [1–5]. While many biological mechanisms have been implicated in the association between lead exposure and increased cardiovascular risk, clinical and experimental evidence is accumulating in support of an important role for lead-induced increase in

*

Corresponding author. Fax: +55 16 633 2301. E-mail address: [email protected] (J.E. Tanus-Santos).

1089-8603/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2005.09.007

oxidative stress and depressed nitric oxide (NO) availability [6–10]. Nitric oxide (NO) plays a pivotal role in the regulation of cardiovascular homeostasis. This highly reactive molecule is produced in endothelial cells and platelets by endothelial NO synthase (eNOS), and it maintains basal vasodilator tone, inhibits platelet aggregation, attenuates leukocyte adhesion to the endothelium, and modulates smooth muscle proliferation [11]. Because of the major importance of eNOS in the regulation of NO availability, many studies addressing the clinical relevance of polymorphisms in the eNOS gene have been carried out [12]. SpeciWcally, three polymorphisms in

F. Barbosa Jr. et al. / Nitric Oxide 14 (2006) 58–64

the eNOS gene have been widely studied: a single nucleotide polymorphism (SNP) in the promoter region (T¡786C), a SNP in exon 7, and the variable number of tandem repeats (VNTR) in intron 4 [12,13]. However, there is only controversial evidence for an impaired NO production as a result of the polymorphism in the exon 7 [14] and no evidence for the VNTR in intron 4 [12]. Conversely, the T¡786C polymorphism reduces the promoter activity by approximately 50% [15,16], thereby lending experimental support to a relevant physiologic role for this SNP. NO is rapidly oxidized to nitrite and nitrate (NOx) in vivo and in vitro. Therefore, measurement of NOx in plasma from blood collected after an overnight fast reXects endogenously produced NO [17–20]. In this regard, we found in healthy volunteers that carriers of the “C” allele for the T¡786C polymorphism tended to have lower NOx concentrations when compared with non carriers of the “C” allele [21]. Indeed, further studies showed that one speciWc eNOS haplotype, which includes the “C,” “4b,” and “Glu” alleles for T¡786C, VNTR in intron 4, and SNP in exon 7, respectively, is associated with lower circulating NOx concentrations [22]. Interestingly, this speciWc eNOS haplotype includes the rarer variant (“C” allele) for the T¡786C polymorphism only, thus suggesting that the T¡786C polymorphism has a more important eVect on NOx concentrations than the other two polymorphisms. While many studies have shown that lead exposure decreases NO availability [6,23], no previous study has examined whether lead exposure aVects plasma NOx concentrations in humans. In addition, while accumulating evidence indicates that the T¡786C polymorphism aVects eNOS expression and endogenous NO release [15,16,24], and maybe the circulating concentrations of NOx [22], no previous study has examined the possible interactions of lead exposure and the genotype for the T¡786C polymorphism. This is important because most common cardiovascular diseases, such as hypertension, depend on the interaction of an individual’s environmental factors with her/his genotype [25], and reduced NOx levels were reported in subjects with essential hypertension compared with normotensives [26]. In this study, we investigated whether there is an association between the circulating concentrations of nitric oxide products (NOx) and the concentrations of lead in whole blood (B-Pb) and in plasma (P-Pb) from lead-exposed subjects. In addition, we also evaluated whether eNOS genotype for the T¡786C polymorphism aVects NOx concentrations in lead-exposed subjects. We hypothesized that lead exposure would decreased circulating NOx concentrations, and that the T¡786C polymorphism would signiWcantly interact with lead exposure and aVect plasma NOx concentrations. Materials and methods Materials High purity de-ionized water (resistivity 18.2 m
cm) obtained by a Milli-Q water puriWcation system (Millipore,

59

Bedford, MA, USA) was used throughout. All reagents used were from high purity analytical grade. All chemical solutions used for Pb determination were stored in highdensity polypropylene bottles. Whole blood and plasma samples were stored in 2 mL tubes at ¡80C. All tubes, plastic bottles, autosampler cups, and glassware materials were cleaned by soaking in 10% v/v HNO3 for 24 h, rinsing Wve times with Milli-Q water and dried in a class 100 laminar Xow hood located inside the class 10,000 clean room. Subjects This study was approved by our institutional review committee, and each subject provided written informed consent. We studied 104 volunteers (33 men and 71 women), aged from 18 to 60, living in the city of Bauru, State of São Paulo, Brazil. Most of them were highly exposed to lead, from air and soil, during the running of a battery plant located near their income area. Although, the battery plant was closed in 2002, part of this population is still exposed, indoor or outdoor, due to constant deposition of lead on soil and vegetation surrounding their houses. In addition, we studied only subjects that were nonsmokers and were not alcohol consumers. Blood collection Venous blood samples were collected from each volunteer after overnight (>12 h) fasting in three separated fractions of 6 mL: two evacuated tubes containing lyophilized heparin (Vacuntainer BD, trace metals free) for metal analysis, and one containing EDTA (Vacuntainer BD) for hematological evaluations and measurement of MMPs activities. Before collection, the skin of the volunteer was cleaned with alcohol and MilliQ water. The Wrst blood fraction was used to determine the B-Pb content and the second was used to plasma collection. All volunteers were asked to come for sample collection after at least 12 h of fasting, since eating has been demonstrated aVect P-Pb concentrations [27]. Blood samples were immediately centrifuged (800g, 6 min) to separate plasma from whole blood, thus avoiding transference of lead from erythrocytes. Each plasma fraction was then pipetted into two ultra-cleaned eppendorVs (2 mL) and immediately frozen at ¡80 °C until used for analysis. Measurement of whole blood and plasma lead concentrations Whole blood samples were analyzed by graphite furnace atomic absorption spectrometry (Varian SpectrAA 220) following the method proposed by Zhou et al. [28] BrieXy, 100 L of blood samples were diluted 1:10 with a solution containing 0.2% v/v HNO3 0.5 Triton X-100. Then, 12 L of the resulted sample was delivered into the graphite tube with graphite platforms previously coated with W-Rh permanent modiWer. Calibration was performed against lead

60

F. Barbosa Jr. et al. / Nitric Oxide 14 (2006) 58–64

aqueous solutions. The method detection limit is 0.7 g/dL. To evaluate the accuracy of the results, NIST 955 whole blood Standard Reference Material and Blood Reference Materials produced by the New York State Department of Health as part of their Interlaboratory Program of ProWciency Testing were analyzed before and after 10 ordinary samples. Plasma samples were analyzed by Inductively Coupled Plasma Mass Spectrometry (Perkin Elmer 6100) following the method proposed by Shutz et al. [29] with modiWcations. BrieXy, 300 L of plasma were diluted 1:10 with a solution containing ammonia (0.04 mol/L), disodium ethylenediaminetetracetic dihydrate (Na2EDTA; 200 mg/L) and Triton X-100 (100 mg/L). The detection limit for lead was 0.001 g/L. Iron levels in plasma were also obtained by ICPMS from each sample to check for hemolysis. Hemolized plasma samples were excluded from the Wnal data. To check the accuracy of the results, Serum Reference Materials produced by the New York State Department of Health as part of their Interlaboratory Program for ProWciency Testing were analyzed before and after 10 ordinary samples. Genotype determination for the T¡786C polymorphism in the 5⬘ Xanking region of eNOS ¡786

Genotypes for the T C polymorphism in the 5⬘-Xanking region of eNOS were determined by polymerase chain reaction (PCR) ampliWcation using the primers 5⬘-TGG AGA GTG CTG GTG TAC CCC A-3⬘(sense) and 5⬘-GCC TCC ACC CCC ACC CTG TC-3⬘(antisense) [13,21,24,30]. The PCR was performed in a 25 l reaction volume that included approximately 100 ng of template genomic DNA, 6.25 pmol of each primer, 200 M of each dNTP, 1.5 mmol/ L MgCl2, 2.5 l of 10£ PCR buVer and 5 U of DNA Taq Polymerase (Biosystems, Curitiba, Brazil). The PCR mixtures were heated to 94 °C for 4 min for denaturation and underwent 35 cycles at 94 °C for 30 s for denaturation, 65 °C for 30 s for annealing, and 72 °C for 1 min for extension. Finally, extension was conducted at 72 °C for 5 min. The ampliWed products were digested with MspI for at least 3 h, at 37 °C, producing fragments of 140 and 40 bp for the wild-type allele (allele “T”), or 90, 50, and 40 bp in the case of a polymorphic variant (allele “C”). Fragments were separated by electrophoresis in 12% polyacrylamide gels and visualized by silver staining (Fig. 1). Measurement of plasma nitrite/nitrate concentrations Venous blood samples were collected in tubes containing EDTA and immediately centrifuged at 1000g for 4 min. Plasma aliquots were then immediately removed and stored at ¡70 °C until analyzed in duplicate for their nitrate content using an ozone-based chemiluminescence assay as previously described [31–33]. BrieXy, the plasma samples were treated with a 2:1 volume of cold ethanol and centrifuged at 14,000g for 5 min. NOx were measured by injecting 25 L of the

140 bp 90 bp

50 bp 40 bp

CC

TT

TC

TC

Genotypes Fig. 1. Genotyping for the T¡786C polymorphism in the promoter region of eNOS gene. The PCR products were digested with restriction enzyme producing diVerent fragments leading to speciWc genotypes.

supernatant in a glass purge vessel containing vanadium (III) in 1 N hydrochloric acid at 90 °C, which reduces nitrite/ nitrate (NOx) to NO gas. A nitrogen stream was bubbled through the purge vessel containing vanadium (III), then through 1 N NaOH, and then into a NO analyzer (Sievers Model 280 NO Analyzer, Boulder, USA), which detects NO released from NOx for chemiluminescent detection. Statistical analysis The distribution of genotypes was assessed for deviation from the Hardy–Weinberg equilibrium by using chi-squared test. The Pearson’s correlation (r, P) was calculated for associations between B-Pb and P-Pb concentrations, NOx concentrations in plasma, and eNOS genotypes. Multiple regression analysis was used to calculate the inter-relationship of all the parameters considered with B-Pb and P-Pb. Because of the relatively low frequency of the CC genotype, we combined both TC and CC genotypes together (TC+CC group) and compared with the TT genotype group. The genotype groups (TT and TC+CC) were evaluated for diVerences in demographic data, B-Pb and P-Pb concentrations, and plasma NOx concentrations by Student’s ttest. In addition, subjects were also classiWed according to their B-Pb concentrations into two groups: low lead concentrations (those subjects with B-Pb concentrations below 100 g/L) and high lead concentrations (those subjects with B-Pb concentrations above 100 g/L), and we carried out a two-way ANOVA (genotype group vs. lead concentration group) to further conWrm an interaction between the genotype and lead exposure. This blood lead concentration (100 g/L) is considered a threshold for concern by the US Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO). P < 0.05 was considered signiWcant throughout. Results Table 1 summarizes the basic characteristics of the study subjects. There were no signiWcant diVerences in the age, gender distribution, body mass index, arterial blood pressure, mean B-Pb and P-Pb concentrations, hematocrit, and NOx concentrations between the genotype groups.

F. Barbosa Jr. et al. / Nitric Oxide 14 (2006) 58–64 Table 1 Demographic characteristics of study participants Variable

Genotypes

N Age (years) Gender (men/women) BMI (kg/m2) SAP (mmHg) DAP (mmHg) HR (bpm) Smokers (%) Alcohol use (%) Blood Pb (g/L) Plasma Pb (g/L) Hematocrit (%) Nitrate/nitrite (M)

TT

TC/CC

35 36.3 § 2.2 11/24 24.3 § 0.7 116.6 § 3.1 73.8 § 1.9 70.8 § 1.7 0 0 70 § 12 0.55 § 0.08 41.8 § 4.1 38.5 § 2.5

69 33.1 § 1.3 22/47 24.5 § 0.4 114.9 § 1.7 73.9 § 1.4 72.4 § 1.1 0 0 64 § 5 0.47 § 0.06 41.0 § 3.7 38.9 § 2.1

Values are the means § SEM. BMI, body mass index; SAP, systolic arterial pressure; DAP, diastolic arterial pressure; HR, heart rate.

No signiWcant correlations were found between the circulating plasma NOx concentrations and B-Pb (Fig. 2A) and P-Pb (Fig. 2B; all P > 0.05) concentrations when the results for all subjects were analyzed. However, while no signiWcant correlation was found between the circulating plasma NOx concentrations and B-Pb (Fig. 3A) and P-Pb (Fig. 3C) in subjects with TT genotype (all P > 0.05), a negative correlation was found between the circulating plasma NOx concentrations and B-Pb (Fig. 3B; r D 0.230, P D 0.048) and P-Pb (Fig. 3D; r D 0.194, P D 0.110) in subjects from TC+CC genotypes group. Finally, the two-way ANOVA showed a signiWcant interaction between the genotype group vs. lead concentration group (F (1, 100) D 4.20, P D 0.043), so that subjects from TC+CC genotype group with B-Pb concentrations above 100 g/L had the lowest circulating plasma NOx concentrations (Fig. 4). Discussion In this study, we demonstrate for the Wrst time that no correlation exists between plasma NOx and B-Pb and P-Pb

concentrations. While these negative results would suggest no eVects of lead exposure on NO availability, subsequent analysis showed that NOx concentrations correlate negatively with B-Pb concentrations in carries of the “C” allele for the T¡786C polymorphism, but not in non carriers of the “C” allele. In addition, although not statistically signiWcant, we found that NOx concentrations also correlated negatively with P-Pb in carriers of the “C” allele. Taken together, these Wndings suggest that lead exposure can aVect NO availability in a select group of subjects carrying the “C” allele. B-Pb concentrations are widely used to diagnose Pb exposure because they may reXect an individual’s current body burden, which is a function of recent and/or past exposure[34]. Indeed, while whole blood Pb measurements are reXective of recent exposure, past exposures may also be represented in them, as a result of Pb mobilization from bone back into blood [35–38]. However, P-Pb may be a more relevant index of exposure, distribution, and health risks associated with lead because the toxic eVects of lead are primarily associated with P-Pb, which reXects the most rapidly exchangeable fraction of lead in the bloodstream [29]. In the present study, we found a signiWcant negative correlation between NOx concentrations and B-Pb in carriers of the “C” allele, and a trend for negative correlation between NOx concentrations and P-Pb. Therefore, independently of which biomarker can better reXect lead exposure, our data strongly suggest that lead exposure may decrease NO availability in carriers of the “C” allele. Finally, we found no diVerences in B-Pb concentrations between eNOS genotype groups, a result consistent with previous data showing no eVect of eNOS genotype for another polymorphism in the eNOS gene on B-Pb concentrations [39]. Our Wndings provide new insight into the biological mechanisms possibly involved in the association between lead exposure and increased cardiovascular risk in some but not all exposed subjects. The signiWcant interaction between lead exposure and the occurrence of the “C” allele may reduce NO availability and increase the susceptibility to cardiovascular diseases. In this regard, it has been shown that the allele “C” of the T¡786C polymorphism signiWB 100

N=104

Nitrite/nitrate (µM)

Nitrite/nitrate (µM)

A 100

61

80 60 40 20 0 0

100

200

300

400

B-Pb (µg/dL)

500

N=104 80 60 40 20 0 0.0

0.5

1.0

1.5

2.0

2.5

P-Pb (µg/L)

Fig. 2. Lack of association between plasma nitrite/nitrate concentrations and whole blood Pb (B-Pb, Panel A) and plasma Pb (P-Pb, Panel B) concentrations in the 104 subjects included in the study.

62

F. Barbosa Jr. et al. / Nitric Oxide 14 (2006) 58–64

A 100

B 100

TT

Nitrite/nitrate (µM)

Nitrite/nitrate (µM)

N=35 80 60 40 20 0

TC+CC r=0.230 P=0.048 N=69

80 60 40 20 0

0

100

200

300

400

500

0

100

Nitrite/nitrate (µM)

Nitrite/nitrate (µM)

D

TT N=35

100

300

B-Pb (µg/L)

B-Pb (µg/L) C

200

80 60 40 20 0

TC+CC r=0.194 P=0.110 N=69

100 80 60 40 20 0

0.0

0.5

1.0

1.5

2.0

2.5

P-Pb (µg/L)

0.0

0.5

1.0

1.5

2.0

2.5

P-Pb (µg/L)

Fig. 3. Lack of association between plasma nitrite/nitrate concentrations and whole blood Pb (B-Pb, Panel A) and plasma Pb (P-Pb, Panel C) concentrations in TT genotype group (N D 35). Negative correlation between plasma nitrite/nitrate concentrations and whole blood Pb (B-Pb, Panel B) and plasma Pb (P-Pb, Panel D) concentrations in TC+CC genotype group (N D 69). The regression line and the 95% conWdence interval are plotted.

Nitrite/nitrate (µM)

50

TT

#

TC+CC TT

TC+CC

40 30 20 10 0

< 100 µg/L

>100 µg/L

Whole blood lead concentration Fig. 4. Plasma nitrite/nitrate concentrations in subjects from TT genotype group (open bars) and from TC+CC genotype group (closed bars), with B-Pb concentrations below 100 g/L and above 100 g/L, respectively. Values are the means § SEM. #P D 0.043, F (1, 100) D 4.20, by two-way ANOVA.

cantly reduces eNOS expression and endogenous NO release [15,16,24]. While, we have reported no signiWcant eVects for this SNP on the circulating NOx concentrations in healthy male subjects[21], our previous study did not have statistical power to detect minor diVerences (less than 30%) in plasma NOx concentrations [21]. In addition, we

did not address possible interactions of the T¡786C polymorphism with environmental factors in that study. The present study, however, provides functional evidence for an important interaction between a genetic marker and an environmental factor. It is possible that carriers of the allele “C” of the T¡786C polymorphism have lower eNOS expression and endogenous NO release [15,16,24], and tend to have lower NOx levels[22], which are further decreased when these subjects are exposed to lead [6–10]. In fact, lead exposure is associated with increased oxidative stress and depressed nitric oxide (NO) availability [6–10], which may be more evident in carriers of the “C” allele. As a limitation of the present Wndings, B-Pb and P-Pb concentrations were relatively small in our study subjects. We could speculate that more signiWcant and stronger correlations would be found if the subjects included in the present study had higher B-Pb and P-Pb concentrations. Moreover, almost all subjects were normotensive in the present study. While it is generally accepted that long-term exposure to lead can cause arterial hypertension, this disease is probably a late complication of lead exposure [6]. Finally, measurement of plasma NOx may not be the best endpoint for determining the eVect of lead exposure on the NOS system. In conclusion, our study shows a negative correlation between plasma NOx concentrations and B-Pb in carriers

F. Barbosa Jr. et al. / Nitric Oxide 14 (2006) 58–64

of the “C” allele, thus suggesting a possible mechanism possibly involved in lead exposure-induced increase in the susceptibility to cardiovascular diseases.

[19]

Acknowledgments This study was supported by Fundação de Amparo à Pesquisa do Estado de Sao Paulo (FAPESP), and Conselho Nacional de Desenvolvimento Cient ´Wco e Tecnológico (CNPq). References [1] Agency for Toxic Substances and Disease Registry, Toxicological ProWle for Lead. Atlanta, Ga: US Department of Health and Human Services, Public Health Services, (1999). [2] L. Moller, T.S. Kristensen, Blood lead as a cardiovascular risk factor, Am. J. Epidemiol. 136 (1992) 1091–1100. [3] A. Navas-Acien, E. Selvin, A.R. Sharrett, E. Calderon-Aranda, E. Silbergeld, E. Guallar, Lead, cadmium, smoking, and increased risk of peripheral arterial disease, Circulation 109 (2004) 3196–3201. [4] J.L. Pirkle, J. Schwartz, J.R. Landis, W.R. Harlan, The relationship between blood lead levels and blood pressure and its cardiovascular risk implications, Am. J. Epidemiol. 121 (1985) 246–258. [5] J. Schwartz, Lead, blood pressure, and cardiovascular disease in men, Arch. Environ. Health 50 (1995) 31–37. [6] N.D. Vaziri, D.A. Sica, Lead-induced hypertension: role of oxidative stress, Curr. Hypertens. Rep. 6 (2004) 314–320. [7] H. Gurer-Orhan, H.U. Sabir, H. Ozgunes, Correlation between clinical indicators of lead poisoning and oxidative stress parameters in controls and lead-exposed workers, Toxicology 195 (2004) 147–154. [8] H. Gurer, N. Ercal, Can antioxidants be beneWcial in the treatment of lead poisoning? Free Radic. Biol. Med. 29 (2000) 927–945. [9] Z. Ni, S. Hou, C.H. Barton, N.D. Vaziri, Lead exposure raises superoxide and hydrogen peroxide in human endothelial and vascular smooth muscle cells, Kidney Int. 66 (2004) 2329–2336. [10] N.D. Vaziri, Y. Ding, EVect of lead on nitric oxide synthase expression in coronary endothelial cells : role of superoxide, Hypertension 37 (2001) 223–226. [11] J.P. Cooke, V.J. Dzau, Nitric oxide synthase: role in the genesis of vascular disease, Annu. Rev. Med. 48 (1997) 489–509. [12] A.D. Hingorani, Polymorphisms in endothelial nitric oxide synthase and atherogenesis: John French Lecture 2000, Atherosclerosis 154 (2001) 521–527. [13] J.E. Tanus-Santos, M. Desai, D.A. Flockhart, EVects of ethnicity on the distribution of clinically relevant endothelial nitric oxide variants, Pharmacogenetics 11 (2001) 719–725. [14] T.A. Fairchild, D. Fulton, J.T. Fontana, J.P. Gratton, T.J. McCabe, W.C. Sessa, Acidic hydrolysis as a mechanism for the cleavage of the Glu298Asp-variant of human endothelial nitric oxide synthase, J. Biol. Chem. 30 (2001) 30. [15] M. Nakayama, H. Yasue, M. Yoshimura, Y. Shimasaki, K. Kugiyama, H. Ogawa, T. Motoyama, Y. Saito, Y. Ogawa, Y. Miyamoto, et al., T-786-C mutation in the 5⬘-Xanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm, Circulation 99 (1999) 2864–2870. [16] Y. Miyamoto, Y. Saito, M. Nakayama, Y. Shimasaki, T. Yoshimura, M. Yoshimura, M. Harada, N. Kajiyama, I. Kishimoto, K. Kuwahara, et al., Replication protein A1 reduces transcription of the endothelial nitric oxide synthase gene containing a ¡786T–C mutation associated with coronary spastic angina, Hum. Mol. Genet. 9 (2000) 2629–2637. [17] M. Rosselli, B. Imthurn, P.J. Keller, E.K. Jackson, R.K. Dubey, Circulating nitric oxide (nitrite/nitrate) levels in postmenopausal women substituted with 17 beta-estradiol and norethisterone acetate. A twoyear follow-up study, Hypertension 25 (1995) 848–853. [18] J.B. Hibbs Jr., C. Westenfelder, R. Taintor, Z. Vavrin, C. Kablitz, R.L. Baranowski, J.H. Ward, R.L. Menlove, M.P. McMurry, J.P. Kushner,

[20]

[21]

[22]

[23]

[24]

[25] [26]

[27] [28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

63

et al., Evidence for cytokine-inducible nitric oxide synthesis from Larginine in patients receiving interleukin-2 therapy, J. Clin. Invest. 89 (1992) 867–877. X.L. Wang, M.C. Mahaney, A.S. Sim, J. Wang, J. Blangero, L. Almasy, R.B. Badenhop, D.E. Wilcken, Genetic contribution of the endothelial constitutive nitric oxide synthase gene to plasma nitric oxide levels, Arterioscler. Thromb. Vasc. Biol. 17 (1997) 3147–3153. C. Baylis, P. Vallance, Measurement of nitrite and nitrate levels in plasma and urine–what does this measure tell us about the activity of the endogenous nitric oxide system? Curr. Opin. Nephrol. Hypertens. 7 (1998) 59–62. S. Nagassaki, I.F. Metzger, D.C. Souza-Costa, A.S. Marroni, J.A. Uzuelli, J.E. Tanus-Santos, eNOS genotype is without eVect on circulating nitrite/nitrate level in healthy male population, Thromb. Res. 115 (2005) 375–379. I.F. Metzger, D.C. Souza-Costa, A.S. Marroni, S. Nagassaki, Z. Desta, D.A. Flockhart, J.E. Tanus-Santos, Endothelial nitric oxide synthase gene haplotypes associated with circulating concentrations of nitric oxide products in healthy men, Pharmacogenomics 15 (2005) 565–570. N.D. Vaziri, K. Liang, Y. Ding, Increased nitric oxide inactivation by reactive oxygen species in lead-induced hypertension, Kidney Int. 56 (1999) 1492–1498. J.E. Tanus-Santos, M. Desai, L.R. Deak, J.C. Pezzullo, D.R. Abernethy, D.A. Flockhart, J.E. Freedman, EVects of endothelial nitric oxide synthase gene polymorphisms on platelet function, nitric oxide release, and interactions with estradiol., Pharmacogenetics 12 (2002) 407–413. C.F. Sing, J.H. Stengard, S.L. Kardia, Genes, environment, and cardiovascular disease, Arterioscler. Thromb. Vasc. Biol. 23 (2003) 1190–1196. K. Node, M. Kitakaze, H. Yoshikawa, H. Kosaka, M. Hori, Reduced plasma concentrations of nitrogen oxide in individuals with essential hypertension, Hypertension 30 (1997) 405–408. W.I. Manton, S.J. Rothenberg, M. Manalo, The lead content of blood serum, Environ. Res. 86 (2001) 263–273. Y. Zhou, R.A. Zanao, F. Barbosa, P.J. Parsons, F.J. Krug, Investigations on a W-Rh permanent modiWer for the detection of Pb in blood by electrothermal atomic absorption spectrometry., Spectrochim. Acta. part B 57 (2002) 1291–1300. A. Schutz, I.A. Bergdahl, A. Ekholm, S. Skerfving, Measurement by ICP-MS of lead in plasma and whole blood of lead workers and controls, Occup. Environ. Med. 53 (1996) 736–740. A.S. Marroni, I.F. Metzger, D.C. Souza-Costa, S. Nagassaki, V.C. Sandrim, R.X. Correa, F. Rios-Santos, J.E. Tanus-Santos, Consistent interethnic diVerences in the distribution of clinically relevant endothelial nitric oxide synthase (eNOS) genetic polymorphisms, Nitric Oxide 12 (2005) 177–182. D.C. Souza-Costa, T. Zerbini, I.F. Metzger, J.B. Rocha, R.F. Gerlach, J.E. Tanus-Santos, L-Arginine attenuates acute pulmonary embolisminduced oxidative stress and pulmonary hypertension, Nitric Oxide 12 (2005) 9–14. M.M. Castro, E. Rizzi, R.R. Rascado, S. Nagassaki, L.M. Bendhack, J.E. Tanus-Santos, Atorvastatin enhances sildenaWl-induced vasodilation through nitric oxide-mediated mechanisms, Eur. J. Pharmacol. 498 (2004) 189–194. X. Wang, J.E. Tanus-Santos, C.D. Reiter, A. Dejam, S. Shiva, R.D. Smith, N. Hogg, M.T. Gladwin, Biological activity of nitric oxide in the plasmatic compartment, Proc. Natl. Acad. Sci. USA 101 (2004) 11477–11482. F. Barbosa Jr., J.E. Tanus-Santos, R.F. Gerlach, P.J. Parsons, Current needs and limitations on the use of biomarkers of internal dose to diagnose lead exposure., Environ Health Perspect (2005) (in press). B.L. Gulson, J.G. Pounds, P. Mushak, B.J. Thomas, B. Gray, M.J. Korsch, Estimation of cumulative lead releases (lead Xux) from the maternal skeleton during pregnancy and lactation, J. Lab. Clin. Med. 134 (1999) 631–640. B.L. Gulson, C.W. Jameson, K.R. MahaVey, K.J. Mizon, M.J. Korsch, G. Vimpani, Pregnancy increases mobilization of lead from maternal skeleton, J. Lab. Clin. Med. 130 (1997) 51–62. B.L. Gulson, K.R. MahaVey, C.W. Jameson, K.J. Mizon, M.J. Korsch, M.A. Cameron, J.A. Eisman, Mobilization of lead from the skeleton

64

F. Barbosa Jr. et al. / Nitric Oxide 14 (2006) 58–64

during the postnatal period is larger than during pregnancy, J. Lab. Clin. Med. 131 (1998) 324–329. [38] E.K. Silbergeld, J. Schwartz, K. MahaVey, Lead and osteoporosis: mobilization of lead from bone in postmenopausal women, Environ. Res. 47 (1988) 79–94.

[39] K. Theppeang, B.S. Schwartz, B.K. Lee, M.E. Lustberg, E.K. Silbergeld, K.T. Kelsey, P.J. Parsons, A.C. Todd, Associations of patella lead with polymorphisms in the vitamin D receptor, delta-aminolevulinic acid dehydratase and endothelial nitric oxide synthase genes, J. Occup. Environ. Med. 46 (2004) 528–537.