Jose E. Tanus-Santos

A Critical Review of Biomarkers Used for Monitoring Human Exposure to Lead: Advantages, Limitations, and Future Needs

Lead concentration in whole blood (BPb) is the primary biomarker used to monitor exposure to this metallic element. The U.S. Centers for Disease Control and Prevention and the World Health Organization define a BPb of 10 μg/dL (0.48 μmol/L) as the threshold of concern in young children. However, recent studies have reported the possibility of adverse health effects, including intellectual impairment in young children, at BPb levels < 10 μg/dL, suggesting that there is no safe level of exposure. It appears impossible to differentiate between low-level chronic Pb exposure and a high-level short Pb exposure based on a single BPb measurement; therefore, serial BPb measurements offer a better estimation of possible health outcomes. The difficulty in assessing the exact nature of Pb exposure is dependent not so much on problems with current analytical methodologies, but rather on the complex toxicokinetics of Pb within various body compartments (i.e., cycling of Pb between bone, blood, and soft tissues). If we are to differentiate more effectively between Pb stored in the body for years and Pb from recent exposure, information on other biomarkers of exposure may be needed. None of the current biomarkers of internal Pb dose have yet been accepted by the scientific community as a reliable substitute for a BPb measurement. This review focuses on the limitations of biomarkers of Pb exposure and the need to improve the accuracy of their measurement. We present here only the traditional analytical protocols in current use, and we attempt to assess the influence of confounding variables on BPb levels. Finally, we discuss the interpretation of BPb data with respect to both external and endogenous Pb exposure, past or recent exposure, as well as the significance of Pb determinations in human specimens including hair, nails, saliva, bone, blood (plasma, whole blood), urine, feces, and exfoliated teeth.

Nitric Oxide Formation Is Inversely Related to Serum Levels of Antiangiogenic Factors Soluble Fms-Like Tyrosine Kinase-1 and Soluble Endogline in Preeclampsia

Deficient NO formation has been implicated in hypertensive disorders of pregnancy. However, no previous study has compared the circulating nitrite concentrations in healthy pregnant women with those found in hypertensive disorders of pregnancy. Moreover, 2 antiangiogenic factors produced in the placenta (soluble fms-like tyrosine kinase-1 and soluble endogline) may affect NO formation during pregnancy. Here, we hypothesized that lower concentrations of markers of NO formation exist in hypertensive disorders of pregnancy and that inverse relationships exist between these markers and soluble fms-like tyrosine kinase-1 or soluble endogline. In this cross-sectional study, we compared 58 healthy pregnant women with 56 gestational hypertensive subjects and 45 preeclamptic patients. We measured plasma and whole blood nitrite concentrations using an ozone-based chemiluminescence assay and serum soluble fms-like tyrosine kinase-1 and soluble endogline concentrations using enzyme immunoassays. Whole blood nitrite levels were significantly lower in gestational hypertensive subjects and preeclamptic patients (−36% and −58%, respectively; both P<0.05) compared with healthy pregnant women. The plasma nitrite levels were ≈37% lower in both groups with hypertensive disorders of pregnancy compared with the group with normotensive pregnancies (both P<0.05). As expected, we found higher circulating soluble fms-like tyrosine kinase-1 and soluble endogline concentrations in preeclampsia compared with gestational hypertensive subjects or with healthy pregnancies (both P<0.05). We found negative correlations between antiangiogenic factors and plasma or whole blood nitrite concentrations (Spearman’s r from −0.175 to −0.226; all P<0.05). Our results show clinical evidence for impaired NO formation in preeclampsia or gestational hypertension. The negative correlations between markers of NO formation and antiangiogenic factors in preeclamptic patients suggest an inhibitory effect for these factors on NO formation.

Antioxidant treatment reduces matrix metalloproteinase-2-induced vascular changes in renovascular hypertension.

Mounting evidence indicates that structural and functional vascular changes associated with two-kidney, one-clip (2K-1C) hypertension result, at least in part, from altered activity of matrix metalloproteinases (MMPs). Because MMPs are upregulated by increased formation of reactive oxygen species (ROS), we hypothesized that antioxidant approaches could attenuate the increases in MMP-2 expression/activity and the vascular dysfunction and remodeling associated with 2K-1C hypertension. Sham-operated or 2K-1C hypertensive rats were treated with tempol 18 mg/kg/day or apocyanin 25 mg/kg/day (or vehicle). Systolic blood pressure was monitored weekly. After 8 weeks of treatment, aortic rings were isolated to assess endothelium-dependent and -independent relaxation. Quantitative morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin sections. Aortic and systemic ROS levels were measured using dihydroethidine and thiobarbituric acid-reactive substances, respectively. Aortic MMP-2 levels and activity were determined by gelatin and in situ zymography, fluorimetry, and immunohistochemistry. Tempol and apocyanin attenuated 2K-1C hypertension (181+/-20.8 and 192+/-17.6 mm Hg, respectively, versus 213+/-18 mm Hg in hypertensive controls; both p<0.05) and prevented the reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Tempol, but not apocyanin (p>0.05), prevented the vascular remodeling found in 2K-1C rats (all p<0.01). Tempol was more effective than apocyanin in attenuating hypertension-induced increases in oxidative stress (both p<0.05), MMP-2 levels, and MMP-2 activity in hypertensive rats (all p<0.05). Our results suggest that antioxidant approaches decrease MMP-2 upregulation and attenuate the vascular dysfunction and remodeling during 2K-1C hypertension.

Circulating Interleukin-6 and High-Sensitivity C-Reactive Protein Decrease After Periodontal Therapy in Otherwise Healthy Subjects

BACKGROUND Periodontal disease has been associated with many chronic inflammatory systemic diseases, and a common chronic inflammation pathway has been suggested for these conditions. However, few studies have evaluated whether periodontal disease, in the absence of other known inflammatory conditions and smoking, affects circulating markers of chronic inflammation. This study compared chronic inflammation markers in control individuals and patients with periodontal disease and observed whether non-surgical periodontal therapy affected inflammatory disease markers after 3 months. METHODS Plasma and serum of 20 controls and 25 patients with periodontal disease were obtained prior to and 3 months after non-surgical periodontal therapy. All patients were non-smokers, they did not use any medication, and they had no history or detectable signs and symptoms of systemic diseases. Periodontal and systemic parameters included probing depth, bleeding on probing, clinical attachment level, hematologic parameters, as well as the following inflammatory markers: interleukin (IL)-6, high-sensitivity C-reactive protein (hs-CRP), CD40 ligand, monocyte chemoattractant protein (MCP)-1, soluble P-selectin (sP-selectin), soluble vascular adhesion molecule (sVCAM)-1, and soluble intercellular adhesion molecule (sICAM)-1. RESULTS There were no differences in the hematologic parameters of the patients in the control and periodontal disease groups. Among the tested inflammatory markers, IL-6 concentrations were higher in the periodontal disease group at baseline compared to the controls (P = 0.006). Therapy was highly effective (P <0.001 for all the analyzed clinical parameters), and a decrease in circulating IL-6 and hs-CRP concentrations was observed 3 months after therapy (P = 0.001 and P = 0.006, respectively). Our results also suggest that the CD40 ligand marker may have been different in the control and periodontal disease groups prior to the therapy (P = 0.009). CONCLUSIONS In apparently otherwise healthy patients, periodontal disease is associated with increased circulating concentrations of IL-6 and hs-CRP, which decreased 3 months after non-surgical periodontal therapy. With regard to the CD40 ligand, MCP-1, sP-selectin, sVCAM-1, and sICAM-1, no changes were seen in the periodontal disease group between baseline and 3 months after therapy.

Sodium nitrite downregulates vascular NADPH oxidase and exerts antihypertensive effects in hypertension.

Dietary nitrite and nitrate are important sources of nitric oxide (NO). However, the use of nitrite as an antihypertensive drug may be limited by increased oxidative stress associated with hypertension. We evaluated the antihypertensive effects of sodium nitrite given in drinking water for 4 weeks in two-kidney one-clip (2K1C) hypertensive rats and the effects induced by nitrite on NO bioavailability and oxidative stress. We found that, even under the increased oxidative stress conditions present in 2K1C hypertension, nitrite reduced systolic blood pressure in a dose-dependent manner. Whereas treatment with nitrite did not significantly change plasma nitrite concentrations in 2K1C rats, it increased plasma nitrate levels significantly. Surprisingly, nitrite treatment exerted antioxidant effects in both hypertensive and sham-normotensive control rats. A series of in vitro experiments was carried out to show that the antioxidant effects induced by nitrite do not involve direct antioxidant effects or xanthine oxidase activity inhibition. Conversely, nitrite decreased vascular NADPH oxidase activity. Taken together, our results show for the first time that nitrite has antihypertensive effects in 2K1C hypertensive rats, which may be due to its antioxidant properties resulting from vascular NADPH oxidase activity inhibition.

Imbalance between matrix metalloproteinases and tissue inhibitor of metalloproteinases in hypertensive vascular remodeling.

Structural vascular changes in two-kidney, one-clip (2K-1C) hypertension may result from increased matrix metalloproteinase (MMP)-2 activity. MMP-2 activation is regulated by other MMPs, including transmembrane-MMPs, and by tissue inhibitors of MMPs (TIMPs). We have investigated the localization of MMP-2, -9, -14, and TIMPs 1-4 in hypertensive aortas and measured their levels by zymography/Western blotting and immunohistochemistry. Gelatinolytic activity was assayed in tissues by in situ zymography. Sham-operated and 2K-1C hypertensive rats were treated with doxycycline (or vehicle) for 8 weeks, and the systolic blood pressure was monitored weekly. Doxycycline attenuated 2K-1C hypertension (165 + or - 11.7 mmHg versus 213 + or - 7.9 mm Hg in hypertensive controls, P<0.01), and completely prevented increase in the thicknesses of the media and the intima in 2K-1C animals (P<0.01). Increased amounts of MMP-2, -9, and -14 were found in hypertensive aortas, as well as enhanced gelatinolytic activity. A gradient in the localization of MMP-2, -9, and -14 was found, with increased amounts detected in the intima, at sites with higher gelatinolytic activity. Doxycycline attenuated hypertension induced increases in all the 3 investigated MMPs in both the media and the intima (all P<0.05), but it did not change the amounts of TIMPs 1-4 (P>0.05). Therefore, an imbalance between increased amounts of MMPs at the tissue level without a corresponding increase in the quantities of TIMPs, particularly in the intima and inner media layers, appears to account for the increased proteolytic activity found in 2K-1C hypertension-induced maladaptive vascular remodeling.

Gingival crevicular fluid levels of MMP‐8, MMP‐9, TIMP‐2, and MPO decrease after periodontal therapy

BACKGROUND This study aimed at comparing the levels of matrix metalloproteinase (MMP)-8, tissue Inhibitor of MMPs (TIMP)-1 and TIMP-2, Myeloperoxidase (MPO), and MMP-9 in the gingival crevicular fluid (GCF) of chronic periodontitis (CP) patients and controls at baseline and 3 months after non-surgical therapy. MATERIALS AND METHODS GCF was collected from one site of 15 control subjects and 27 CP patients. MMP-8, MMP-9, TIMP-1, and TIMP-2 were determined by Enzyme-linked immunoabsorbent assay; different forms of MMP-9, by gelatin zymography; and MPO, colorimetrically. RESULTS At baseline, higher levels of MMP-8, TIMP-2, MPO, and the 87 kDa-MMP-9 were found in patients compared with controls (p<0.001), and these molecules decreased after therapy (p<0.03). There were no differences between the groups with respect to the higher molecular forms of MMP-9 (180, 130, 92 kDa) or total MMP-9 at baseline. No differences were observed in TIMP-1 levels. In controls, decreased levels of TIMP-2 and the higher molecular forms of MMP-9 (180, 130, 92 kDa) were found 3 months after therapy compared with baseline (p<0.01). CONCLUSIONS Higher levels of MMP-8, TIMP-2, MPO, and 87 kDa MMP-9 were found in the GCF of patients compared with controls, and these markers decreased 3 months after periodontal therapy.

Comparative assessment of matrix metalloproteinase (MMP)-2 and MMP-9, and their inhibitors, tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in preeclampsia and gestational hypertension

OBJECTIVES To compare the circulating levels of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2, and the MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios in preeclampsia and gestational hypertension with those found in normotensive pregnancies. DESIGN AND METHODS We studied 83 pregnant women (30 healthy pregnant women with uncomplicated pregnancies, 26 with gestational hypertension, and 27 with preeclampsia) and 30 healthy nonpregnant women in a cross-sectional study. MMP and TIMP concentrations were measured in plasma samples by gelatin zymography and ELISA, respectively. RESULTS We found higher plasma pro-MMP-9 levels, and higher pro-MMP-9/TIMP-1 ratios in women with gestational hypertension (95%-CI: 1.031 to 2.357, and 0.012 to 0.031, respectively), but not with preeclampsia, compared with those found in normotensive pregnant women (95%-CI: 0.810 to 1.350, and 0.006 to 0.013, respectively; both P<0.05). We found no significant differences in pro-MMP-2 levels (P>0.05). CONCLUSIONS The higher net MMP-9 (but not MMP-2) activity in gestational hypertension compared with normotensive pregnancy suggests that MMP-9 plays a role in the pathophysiology of gestational hypertension. Conversely, the lack of such alterations in preeclampsia is consistent with the notion that different pathophysiological mechanisms are involved in these hypertensive disorders.

The effect of sildenafil on pulmonary embolism-induced oxidative stress and pulmonary hypertension.

Acute pulmonary embolism (APE) is a major cause of pulmonary hypertension and death. We examined the effects of sildenafil on the hemodynamic changes caused by APE in anesthetized dogs. Sham-operated dogs (n = 3) received only saline. APE was induced by stepwise IV injections of 300 &mgr;m microspheres in amounts adjusted to increase mean pulmonary artery pressures by 20 mm Hg. Hemodynamic evaluation was performed at baseline, after APE was induced, and then after sildenafil 0.25 mg/kg (n = 8), or sildenafil 1 mg/kg + 0.3 mg · kg−1 · h−1 (n = 8) or saline (n = 9) infusions were started. Similar experiments were conducted to examine the effects of sildenafil in rat isolated perfused lung preparation. Plasma thiobarbituric acid reactive species were also determined in both studies to measure oxidative stress. Both doses of sildenafil reduced mean pulmonary artery pressures in dogs by approximately 8 to 16 mm Hg (both P < 0.05) and attenuated the increase in oxidative stress after APE. Mean arterial blood pressure remained unaltered after both doses of sildenafil. Sildenafil produced similar effects after APE in rat isolated perfused lung preparation. These findings indicate that IV sildenafil can selectively attenuate the increases in mean pulmonary artery pressures after APE, possibly through antioxidant mechanisms.

Increased circulating levels of matrix metalloproteinase (MMP)-8, MMP-9, and pro-inflammatory markers in patients with metabolic syndrome

BACKGROUND Metabolic syndrome (MetS) predisposes to cardiovascular complications. Increased concentrations of pro-inflammatory mediators and imbalanced concentrations of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) may reflect the pathophysiology of MetS. We compared the circulating levels of MMPs, TIMPs, and inflammatory mediators in MetS patients with those found in healthy controls. METHODS We studied 25 healthy subjects and 25 MetS patients. The plasma levels of pro-MMP-2 and pro-MMP-9 were determined by gelatin zymography. The plasma concentrations of MMP-8, MMP-3, TIMP-1, TIMP-2, monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), intercellular adhesion molecule (sICAM-1), and sP-selectin were measured by ELISA kits. RESULTS We found higher sP-selectin, sICAM-1, MCP-1, and IL-6 (all P<0.05) concentrations in MetS patients compared with healthy controls. No differences in pro-MMP-2, MMP-3, and TIMP-2 levels were found (all P>0.05). However, we found higher pro-MMP-9, MMP-8, and TIMP-1 levels in MetS patients compared with healthy controls (all P<0.05). CONCLUSIONS Patients with MetS have increased circulating concentrations of pro-MMP-9, MMP-8, and TIMP-1 that are associated with increased concentrations of pro-inflammatory mediators and adhesion molecules. These findings suggest that MMPs may have a role in the increased cardiovascular risk of MetS patients. Pharmacological interventions targeting MMPs, especially MMP-9 and MMP-8 deserve further investigation in MetS patients.