Early studies by our group have shown that lead-induced hypertension (HTN) is closely related to enhanced activity of reactive oxygen species (ROS). In addition, we have found indirect evidence that hydroxyl radical may be the most likely culprit in lead-exposed animals. In the present study, rat aortic endothelial cells were incubated in the presence of 0, 0.01, 0.1, 0.5, and 1.0 ppm lead acetate for 1, 24, and 48 h. At the conclusion of the incubation period cells were harvested and the media were collected. Lipid peroxidation products were measured as malondialdehyde-thiobarbituric acid (MDA-TBA) in the medium and hydroxyl radical was measured as 2,3-dihydroxybenzoic acid (2,3 DHBA) in the cells. After exposure to lead for 48 h, MDA-TBA generation and 2,3 DHBA formation were significantly increased. These data clearly demonstrate that lead exposure promotes hydroxyl radical generation and induces oxidative stress in isolated endothelial cells, mimicking the effects observed in lead-exposed animals. Enhanced inactivation of endothelium-derived nitric oxide by locally produced oxygen free radicals could contribute to endothelial dysfunction and HTN in lead-exposed animals.
Oral administration of low levels of lead for 3 months has been shown to cause severe sustained hypertension (HTN) in genetically normotensive rats,1,,,,–6 without affecting renal function or structure.7 In a series of studies, we have demonstrated that lead-induced HTN is associated with increased reactive oxygen species (ROS) in this model.3,–5,6 This supposition has been based upon the finding of increased lipid peroxidation4,–6 and tissue nitrotyrosine abundance, which are footprints of excess ROS activity.6 We hypothesized that increased ROS activity contributed to HTN by enhancing inactivation of endothelium-derived nitric oxide. In support of this hypothesis, we have shown that correction of oxidative stress by certain antioxidants results in amelioration of HTN and enhanced nitric oxide (NO) availability in lead-exposed rats.4,6 In an earlier study, we showed normalization of blood pressure and urinary excretion of NO metabolites with the reputed hydroxyl radical scavenger, dimethylthiourea (DMTU) (unpublished data). However, neither superoxide dismutase (SOD), a superoxide scavenger enzyme, nor catalase, a hydrogen peroxide scavenging enzyme, significantly affected these parameters.5 These observations indirectly pointed to hydroxyl radical as the most likely culprit in this model. The present study was undertaken to test the hypothesis that lead exposure enhances hydroxyl radical generation. To this end, rat aortic endothelial cells were studied in vitro to isolate the direct effect of lead from those associated with hemodynamic and other systemic actions of lead that are inherent to the intact animal.
Materials and methods
Adult Sprague-Dawley rats were anesthetized by an intraperitoneal injection of nembutal (50 mg/kg), and a thoracotomy was performed. The full length of the thoracic aorta was sterilely removed, rinsed three times with PBS, and placed into a 100-mm culture dish filled with serum-free culture medium on ice. The vessel was gently cleaned of periadventitial fat and connective tissue, cut to expose the luminal surface, and rinsed with the culture medium. The surface was then covered with collagenase solution (II and IV, 1 mg/mL) and incubated at 37°C for 1 h. After incubation, the solution containing detached endothelial cells was aspirated and placed into a tube with 5 mL medium plus serum to arrest the digestion process. The vessel surface was then subjected to a forceful stream of the culture medium using a 10-mL syringe to collect the remaining cells loosely attached to the surface. Finally, the vessel was rinsed with the medium, and the medium containing additional cells was collected and combined with the initial aspirate. The cell suspension was centrifuged at 2000 rpm for 10 min. The cell pellet was washed twice, suspended in medium to a total volume of 2 mL, and placed into a 60-mm culture dish. The dish was then placed in a humidified incubator at 37°C with 5% CO2. After 2 days, 2 mL of fresh medium was added to the dish, and the incubation was continued for an additional 2 days. Thereafter, the medium was changed every other day. Once the cells formed a monolayer, the cells were subculture. The cells were identified by staining with a specific antibody to von Willebrand factor and fluorescent-labeled LDL, as described previously.8 The Minimum Essential Medium Eagle (MEM), D-Valine modification (Sigma Chemical Inc., St Louis, MO), was used in these experiments. Fetal calf serum, penicillin, streptomycin endothelial cell growth supplement, and heparin (Sigma Chemical Inc.) were used as appropriate. Distilled water was used and strict aseptic measures were observed in all experiments to avoid bacterial and endotoxin contaminations.
The cells obtained on passages 4 and 5 were used for the experiments. Once 70% to 80% confluence was reached, the cells were treated with either 0.01, 0.1, 0.05, and 1.0 ppm lead acetate or inactive vehicle in a medium containing 5% fetal calf serum for 1, 24, and 48 h. At the conclusion of each treatment period, the cells and the supernatants were harvested and stored at −70°C until processed.
Determination of lipoperoxides
Lipoperoxides in the medium were determined by measurement of malondialdehyde-thiobarbituric acid (MDA-TBA), using HPLC, as described earlier.4
Determination of hydroxyl free radicals
The cells, treated with various concentrations of lead or vehicle, were suspended in 0.5 mL of serum-free culture medium to which 2 mmol/L sodium salicylate (pH 7.4) was added. The cells were then incubated at 37°C for 80 min. After incubation, the cells were pelleted by centrifugation, and then 0.4 mL of 15% trichloroacetic acid (TCA) was added to the pellet fraction, which was subsequently centrifuged at 3000 g for 20 min. The supernatant was collected and analyzed for 2,3-dihydroxybenzoic acid (DHBA), 2,5 DHBA, and salicylate using HPLC, as described previously.9
Data presentation and analysis
Analysis of variance (ANOVA) and Student's t test were used in statistical evaluation of the data, which are given as mean ± SEM. P values less than .05 were considered significant.
Exposure of cultured rat aortic endothelial cells for 48 h to lead acetate at 0.1 ppm, 0.5 ppm, and 1.0 ppm resulted in significant increases in formation of malondialdehyde (MDA). Similarly, a significant rise in MDA generation was noted in cells incubated with lead acetate for 24 h at 1.0 ppm concentration. These observations point to induction of oxidative stress in cultured endothelial cells by lead. Data are illustrated in Figure 1.
Generation of lipid peroxidation product, malondialdehyde (MDA), in cultured rat aorta endothelial cells incubated with either inactive vehicle or lead acetate at the given concentrations for 1, 24, and 48 h. *P < .05, compared with control (Pb 0 ppm); **P < .01, compared with control (Pb 0 ppm).
Hydroxyl radical production
Data are shown in Figure 2. A significant increase in 2,3-DHBA production was noted in endothelial cells incubated with lead acetate at 1.0 ppm for 48 h. This observation points to enhanced hydroxyl radical generation in lead-treated endothelial cells. No discernible increase was observed with shorter incubation periods. In addition, salicylate and 2,5-DHBA levels remained relatively constant.
Hydroxyl radical production expressed as 2,3-DHBA in response to incubation with lead acetate at the given concentrations for 48 h. **P < .01.
Cell viability as discerned from trypan blue exclusion test was greater than 90% for all experiments and similar in the lead-treated and untreated cells. Similarly, cell count and cellular total protein contents were unaffected by the given treatments. Data are depicted in Figure 3.
Protein content of lysed endothelial cells and cell count after incubation with 1.0 ppm of lead acetate for 1, 24, and 48 h.
Exposure to lead acetate concentrations ranging between 0.1 and 1.0 ppm resulted in a marked increase in lipid peroxidation, as discerned from MDA measurements. Thus, lead acetate at the given concentrations promoted oxidative stress in isolated endothelial cells. The results of the present in vitro studies are in conformity with those of our earlier in vivo studies, which revealed increased MDA generation in lead-treated rats.3,–5 The fact that lead exposure resulted in oxidative stress in isolated endothelial cells in vitro points to the direct action of lead on cellular metabolism. Consequently, oxidative stress seen in the lead-treated animals appears to be a primary event, as opposed to being a consequence of the associated HTN. This viewpoint is supported by the observation that the rise in plasma MDA in the lead-treated animals precedes the rise in arterial blood pressure.6
We have previously shown that administration of DMTU, a reputed hydroxyl radical scavenger, resulted in rapid reduction of blood pressure to normal values in rats with lead-induced HTN but had no discernible effect in normal animals (unpublished data). Similarly, we have shown a significant response to DMTU administration in rats with uremic hypertension.10 These observations pointed to increased hydroxyl radical activity in these models. This supposition was confirmed by the results of the present study that demonstrated enhanced hydroxyl radical generation by lead exposure in cultured endothelial cells.
Endothelium-derived NO is the most potent endogenous vasodilator and as such plays a major role in blood pressure homeostasis. Hydroxyl radicals and other ROS avidly react with and inactivate NO.11 Therefore, enhanced generation of hydroxyl radicals in lead-treated endothelial cells necessarily results in enhanced oxidation and inactivation of locally produced NO. This can, in turn, contribute to the genesis of the associated HTN by disturbing the balance between the vasodilatory and vasoconstrictive factors. Additionally, either superoxide or hydroxyl radical may affect blood pressure directly. In this regard, Katusic and Vanhoutte12 have shown that superoxide causes contraction of canine basilar arteries in vitro. Similarly, Auch-Schwelk et al13 have demonstrated that blood vessels from spontaneously hypertensive rats exhibit concentration-dependent vascular contractions on exposure to xanthine plus xanthine oxidase-generated superoxide. Interestingly, deferoxamine, the iron chelator that prevents generation of hydroxyl radical, totally abolished the contractions. The latter phenomenon pointed to hydroxyl radical as the cause of vasoconstriction in this system.13
Reactive oxygen species, particularly hydroxyl radical, are known to raise cytosolic Ca2+ concentration ([Ca2+]i) in different cell types.14,–16 This effect can be augmented by ROS-medicated inactivation of NO, which exerts its vasodilatory action via a cGMP-stimulated lowering of [Ca2+]i in vascular smooth muscle cells. These events may work in concert to raise [Ca2+]i (which is the common mediator of vasoconstriction) in lead-exposed animals.
In conclusion, exposure to lead promoted hydroxyl radical generation and lipid peroxidation in cultured rat endothelial cells. Enhanced inactivation of endothelium-derived NO by locally produced oxygen free radicals can contribute to endothelial dysfunction and HTN in lead-exposed animals. Further studies are required to explore the effects of specific ROS scavengers on oxidation-induced events in lead-treated endothelial cells.