November 2012

Biol. Pharm. Bull. 35(11) 18851891 (2012)

1885

Current Topics

Advances in Biology and Toxicology of Environmental Metals/Metalloids Cellular Defense Mechanisms against Lead Toxicity in the Vascular System
Yasuhiro Shinkaia and Toshiyuki Kaji*,b
Environmental Medicine Section, Faculty of Medicine, University of Tsukuba; 111 Tennodai, Tsukuba, Ibaraki 3058575, Japan: and b Department of Environmental Health, Faculty of Pharmaceutical Sciences, Tokyo University of Science; 2641 Yamazaki, Noda, Chiba 2788510, Japan. Received June 14, 2012 Lead is a toxic heavy metal that can cause a range of health problems. In this context, the vascular system is a particular target of the deleterious effects of lead. Lead exerts its toxicity through substitution of other divalent cations such as calcium and zinc, resulting in disruption of homeostasis. Based on the evidence that lead up-regulates endoplasmic reticulum (ER) chaperone glucose-regulated protein 78 (GRP78) and/or antioxidant proteins such as hemeoxygenase-1, it is believed that the heavy metal is able to induce ER and/ or oxidative stress in cells. These events also suggest that the unfolded protein response (UPR) system and the antioxidant defense system Kelch-like ECH-associated protein 1nuclear factor (NF)-E2-related factor 2 (Keap1Nrf2) play a critical role in adaptive response to lead. In this review, we summarize recent progress in lead toxicity in terms of cellular defense systems, including stress proteins and transcription factors involved in the vascular system. Key words lead; glucose-regulated protein 78; unfolded protein response; Kelch-like ECH-associated protein 1; nuclear factor-E2-related factor 2
a

1.

INTRODUCTION

Lead is a common environmental and occupational contaminant released from natural and anthropogenic sources. Inorganic lead can be absorbed through the respiratory tract via inhalation of lead dust and lead containing paints and also through the gastrointestinal tract from water and food contaminated by lead content in the soil.1) After absorption, lead is distributed in the blood, bone, and soft tissues.2) Because lead is a cumulative heavy metal, the half-life of lead in blood is 35 d, in brain it is about 2 years, and in bone it persists for decades.3) It is well documented that lead can cause adverse health effects including dysfunctions of nervous, renal, and vascular systems.4) In particular, lead exposure has been shown to be an important determinant of blood pressure levels and hypertension in human population studies.5) Lead challenge has also been associated with an increased incidence of cardiovascular disorders such as coronary heart disease, stroke, and peripheral arterial disease.6,7) To elucidate the molecular basis for the symptoms of lead toxicity observed in humans, extensive studies with different cultured cells have been conducted over the past two decades. Although the mechanisms underlying lead toxicity are still a matter of research, it has been suggested that the toxicity of lead is mainly owing to its ability to substitute for other polyvalent cations (particularly divalent cations, such as calcium [Ca 2+] and zinc [Zn2+]) in the molecular machinery of living organisms.8) Also, accumulating evidence indicates that endoplasmic reticulum (ER) stress and oxidative stress could, in part, underlie some of the toxic effects of lead.9,10) Consistent
* To whom correspondence should be addressed. e-mail: t-kaji@rs.tus.ac.jp

with this notion, some of the gene products induced by lead act as ER-resident molecular chaperones or antioxidant proteins (Table 1); this can be accounted for by an adaptive response to lead. In this review, we discuss cellular defense mechanisms against lead in relationship to the toxicity of lead in the vascular system.

2.

VASCULAR DYSFUNCTION BY LEAD

Numerous population studies have demonstrated a link between long-term lead exposure and elevated arterial blood pressure.2) This epidemiologic relationship is also supported by experimental and mechanistic evidence. Several studies with experimental animals have suggested that chronic lead exposure is linked to hypertension and atherosclerosis.2,11) The effects of lead on vascular function have been explored in in vivo and in vitro studies. The endothelium plays a central role in regulation of vascular function and endothelial damage or dysfunction results in atherosclerosis, thrombosis, and tissue injury. We have reported that lead exhibits adverse effects on vascular endothelial and smooth muscle cells.12) Using cultured endothelial cells, we showed that lead nitrate inhibits the repair of wounded monolayers (also shown in Fig. 1), reduces proliferation, and attenuates growth stimulated by zinc without non-specic cell damage.1315) In addition, lead has been shown to interfere with the synthesis of glycosaminoglycans and heparan sulfate proteoglycan in cultured bovine aorta endothelial cells (BAEC).16) This was accompanied by a marked reduction of the core protein of perlecan, which is a high-molecular-weight heparan sulfate proteoglycan.17)
2012 The Pharmaceutical Society of Japan

1886 Table 1. Stress Proteins/Genes Induced by Lead Exposure 1 M for 7 d Lead nitrate (12.5100 M) for 48 h Lead acetate (1 M) for 1 week Lead nitrate (50 g/mL) for 48 h Lead chloride (60300 M) for 24 h Lead nitrate (225 M) for 1224 h Lead nitrate (225 M) for 1224 h Lead nitrate (50 g/mL) for 48 h Lead acetate (50 mg/kg) for 324 h Pb2+ (100 nM10 M) for 1 d Lead nitrate (25 M) for 6 h Lead chloride (50 M) for 48 h Lead nitrate (2550 M) for 24 h Lead nitrate (2550 M) for 24 h Lead nitrate (25 M) for 6 h Lead chloride (5 or 50 M) for 48 h Lead nitrate (50100 M) for 48 h Lead nitrate (25 M) for 6 h Lead (25 M) for 24 h Lead acetate (300 mol/kg) for 424 h Lead chloride (50 M) for 48 h Lead (1 ppm) for 24 h Lead nitrate (1 M) for 1 h Lead acetate (0.110 M) for 24 h Lead acetate (1050 mg/kg) for 4 weeks Lead acetate (25 mg/kg) for 3 d Source C6 rat glioma cells HepG2 cells Rat primary astroglia HepG2 cells NRK-52E cells BAEC BAEC HepG2 cells Rat kidney cortex Rat hippocampal astrocytes Hepa1c1c7 cells HUVEC HAEC HAEC Hepa1c1c7 cells HUVEC HepG2 cells Hepa1c1c7 cells Rat mesencephalic cells Mouse liver/kidney HUVEC Rat aortic segments A431 cells PC12 cells Mouse liver Rat brain

Vol. 35, No. 11

Protein/gene name GRP78

Reference number 31, 32 36 10 35 34 33 33 35 40 38 39 41 This paper This paper 42, 43 41 36 42 44 45 41 47 46 48 49 50

GRP94 CHOP HO-1

GCLM NQO1 GSTA1 MT HSP70 COX-2 Bax GFAP

Perlecan contributes to endothelial cell growth and repair processes by facilitating broblast growth factor-2 binding to its receptor on endothelial cells. Considering the importance of perlecan function, its down-regulation by lead may adversely affect endothelial repair and promote atherosclerosis. Contrary to the inhibitory effect on endothelial cell growth, lead has been shown to stimulate proliferation of vascular smooth muscle cells in a concentration-dependent manner,18) suggesting that lead is potentially involved in the evolution of atherosclerosis by multiple mechanisms. Alternatively, prevention of intravascular coagulation and preservation of blood uidity are also important functions of the endothelium. Fibrinolysis in normally circulating blood strongly depends on the balance between tissue plasminogen activator (t-PA) and PA inhibitor-1 (PAI-1); t-PA is bound to brin and converts plasminogen to plasmin that can degrade brin. We have also found that lead causes a signicant inhibition of t-PA release in human umbilical vein endothelial cells (HUVEC) as well as in human aorta smooth muscle cells.19) Moreover, lead chloride increases PAI-1 release in human broblastic IMR-90 cells.20) Therefore, lead exposure may increase the risk of thrombosis.

Fig. 1. Lead Inhibits the Repair of Wounded Monolayers of Human Aortic Endothelial Cells (HAEC)
Dense cultures of cells pretreated with 10 or 50 M lead nitrate (Pb) for 24 h were damaged with a cell scraper and then incubated for 12 h in the presence of Pb (10 or 50 M) in HuMedia EG-2. Under these conditions, nonspeci c cell damage did not occur (data not shown). Morphology of the wounded area of HAEC monolayers was observed by phase-contrast microscopy (original magni cation 40). Scale bar=250 m.

3. POSSIBLE MECHANISMS UNDERLYING (VASCULAR) TOXICITY OF LEAD

Lead can enter cells by several pathways such as Ca2+ channels or divalent metal transporter 1 (DMT1), and can be distributed in various cellular destinations including organelles, proteins, and DNA. A current explanation for lead toxicity is that it can substitute for diverse endogenous cations such as calcium and zinc because of its ability to interact with

oxygen and sulfur, both of which relate to protein metal-binding sites. Because lead has a larger ionic radius and greater electronegativity compared to zinc and calcium, it exhibits favorable interactions with coordinating groups of proteins. In contrast to endogenous cations, the distribution of the electrical charge around lead is irregular because of the presence of an inert electron pair in its electronic cloud, affecting protein structure and/or function.8) It has been reported that lead has an inhibitory effect on zinc-binding proteins, whereas lead can cause abnormal activation of several calcium-binding proteins.3) For example, lead exposure stimulates protein kinase C (PKC), calmodulin, and cAMP phoshodiesterase.2123) Because calmodulin is involved in the modulation of several ionic channels, lead not only affects cell signaling pathways by replacing calcium in protein binding sites, but also alters

November 2012

1887

calcium concentration by modulating the activity of ion channels. The ER is one of the main reservoirs of calcium in the cell. It was reported that lead inhibits Ca2+-ATPase activity, increasing the cytoplasmic concentration of calcium with a consequent reduction in ion concentration inside the ER.24) Because many signaling and chaperone proteins inside the ER are calcium-dependent, calcium depletion from this organelle leads to the induction of ER stress. In addition, lead can bind directly to a calcium-binding protein, glucose-regulated protein 78 (GRP78), which is an ER-resident molecular chaperone, and induce its aggregation.25) Lead is also a potent protein folding inhibitor in vitro.26) Therefore, it is likely that lead causes ER stress via multiple mechanisms such as disruption of calcium homeostasis, inactivation of GRP78, and inhibition of protein folding in the ER. Although lead is considered to be a poor inducer of oxidative stress, the inhibition of several antioxidant enzymes such as superoxide dismutase (SOD), catalase, glutathione (GSH) peroxidase, and GSH reductase concomitant with increased lipid peroxidation has been reported.9) In this context, different mechanisms could be involved in lead-induced pro-oxidant action.27) Lead can directly bind to protein and non-protein thiol groups, thereby decreasing the reductive potency of antioxidant enzymes and GSH. Also, lead may induce oxidative stress by inhibiting the enzyme, delta-aminolevulinic acid dehydratase, which results in the accumulation of deltaaminolevulinic acid that can be rapidly oxidized to generate reactive oxygen species (ROS). Furthermore, PKC activation may be involved in lead-induced oxidative stress because PKC can trigger the activation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex, resulting in production of ROS.28) It has been shown that lead interacts with negatively charged phospholipids in membranes, thereby inducing changes in the physical properties of membranes and subsequent propagation of lipid peroxidation.29) Lead can also increase the oxidizing potential of oxidant species by forming lead-superoxide complexes with higher oxidizing capacity.29)

4. STRESS PROTEINS AND GENES INDUCED BY LEAD

The elevated expression of stress proteins is considered to be an adaptive response to adverse conditions. Stress proteins induced by lead, which represent a potential mechanism of cellular defense against lead, are summarized in Table 1. Both GRP94 and GRP78 have been identied as calcium-binding proteins.30) These ER-resident molecular chaperones play important roles in protein folding/assembly and ER calcium binding. These proteins are induced by disruption of ER function, thereby acting as ER stress markers. It has been reported that lead induces GRP78 and GRP94 expression in several cell lines including vascular endothelial cells.10,3136) Knockdown of GRP78 results in an increase in the generation of ROS and cytotoxicity by lead,33,37) suggesting that ER chaperones function as a defense mechanism against lead toxicity. CHOP (another ER stress marker protein, also termed GADD153) is a transcription factor involved in ER stress-induced apoptosis. Tchounwou et al. reported that lead increases CHOP promoter activity in HepG2 cells.35) Induction of these ER stress-associated proteins by lead suggests that ER stress

response plays an important role in handling lead toxicity. Heme oxygenase-1 (HO-1) is a major inducible stress protein associated with protection against oxidative stress. It has been shown that lead induces HO-1 in a number of cell lines including vascular endothelial cells (also shown in Fig. 2).3841) Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the glutathione biosynthesis pathway. GCL is a heterodimer consisting of a catalytic subunit (GCLC) and a modier subunit (GCLM). As shown in Fig. 2, lead induces GCLM expression in a concentration-dependent manner in human aortic endothelial cells (HAEC). GSH S -transferase (GST) belongs to a family of enzymes that catalyze the formation of the thiol group of glutathione to electrophilic xenobiotics. Lead can upregulate GSTA1 in Hepa1c1c7 and HepG2 cells.36,42) NADPH: quinone oxidoreductase 1 (NQO1) is a avoprotein that catalyzes the two-electron reduction of quinone compounds and thus prevents the generation of ROS derived from the interaction of the semiquinone with molecular oxygen. It has been reported that lead increases NQO1 in Hepa1c1c7 cells and HUVEC.4143) Because antioxidant and detoxifying proteins such as HO-1, GCLM, GSTA1, and NQO1 are regulated by transcription factor Nrf2, induction of these proteins by lead indicates that the Kelch-like ECH-associated protein 1nuclear factor-erythroid 2-related factor 2 (Keap1Nrf2) system contributes to the protective response against lead toxicity. Besides the above-mentioned proteins, lead can induce metallothionein (MT),44,45) heat shock protein 70 (HSP70),41) pro-in ammatory protein cyclooxygenase-2 (COX-2),46,47) pro-apoptotic protein Bax,48,49) and glial brillary acidic protein (GFAP).50) GFAP has been recently identied as a regulator of chaperone-mediated autophagy,51) suggesting that the induction of GFAP serves to degrade aggregated proteins resulting from lead exposure. Several researchers have also conducted microarray analyses to study the mechanisms of stress response to lead. Bouton et al. identied calcium-dependent phospholipid binding protein, annexin A5, as a lead-induced protein in astrocytes by using microarray analysis.52) Pan et al. reported that GRP78, K14, -actin, and RhoGDI2 were lead-induced proteins in mice skin.53) Although lead can induce a variety of stress proteins, it appears that the ER stress response system and the Keap1Nrf2 system play a central role in protection against lead toxicity, especially in the vascular system.

5.

UNFOLDED PROTEIN RESPONSE (UPR) TO LEAD

ER dysfunction causes accumulation of unfolded proteins in the ER and triggers an ER stress response termed the UPR. Activation of the UPR alleviates ER stress by decreasing protein synthesis and increasing the protein folding and degradation pathways, leading to cellular recovery. However, programmed cell death signaling pathways are also induced by UPR and the balance of these responses determines cellular survival or death. The three branches of the UPR are well established.54,55) Three families of signal transinducers (PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1)) sense the presence of unfolded proteins in the ER lumen and transmit signals into the nucleus. As shown in Table 1, lead induces UPR target genes such as GRP78, GRP94, and CHOP in a variety of cell types, indicating that one or more UPR pathways

1888

Vol. 35, No. 11

PERK pathway can be activated by lead-induced ER stress. ATF6 is a membrane-bound transcription factor activated by proteolysis in response to ER stress to induce ER chaperone genes such as GRP78. Our recent report demonstrated that although ATF6 is partially involved in basal expression of GRP78 and GRP94, ATF6 does not contribute to the inducible expression of GRP78 and GRP94 by lead exposure in BAEC.33) Under the same conditions, lead-induced cleavage of ATF6 is not observed, suggesting that the ATF6 pathway is not activated by lead-induced ER stress. IRE1 is a bifunctional transmembrane kinase/endoribonuclease that induces the unconventional splicing of XBP1 mRNA to produce the active form of transcription factor XBP1s. This transcription factor regulates several UPR target genes including ER chaperones and ER-associated protein degradation components. In addition to the splicing of XBP1 mRNA, IRE1 kinase can also activate the c-Jun N-terminal kinase (JNK) signaling pathway. We have found that lead increases phosphorylation of IRE1 and JNK in BAEC.33) Also, lead-induced GRP78 and GRP94 expression is completely blocked by the JNK inhibitor, SP600125, or activator protein-1 (AP-1) inhibitor, curcumin, suggesting that lead induces the expression of GRP78 and GRP94 via the activation of the JNK-AP-1 pathway but not XBP1 in endothelial cells.

6.
Fig. 2. Lead Activates Nrf2 and Its Downstream Proteins in HAEC
(A) Cells were exposed to 10 M Pb for 1, 3, 6, 12, 24 h. (B) Cells were exposed to Pb (5, 10, 25, or 50 M) for 24 h. Total cell lysates were subjected to Western blot analysis as described in Shinkai et al.33) with the indicated antibodies. Anti-Nrf2 (H-300) antibody and anti-GCLM (FL-274) antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Anti-HO-1 antibody was obtained from Stressgen (Victoria, Canada). The intensity of the bands was measured using densitometry. Each value represents the meanS.E. (n=3). * p<0.05 or ** p<0.01 compared with control.

THE KEAP1NRF2 SYSTEM

are activated in response to lead. PERK is a transmembrane kinase that phosphorylates translation initiation factor eIF2, thereby reducing cellular protein synthesis. Alternatively, eIF2 phosphorylation also allows the translation of some mRNAs that contain small open reading frames in the 5-untranslated regions, such as transcription factor ATF4. An important target gene of ATF4 is a transcription factor CHOP that controls genes encoding components involved in apoptosis. As shown in Fig. 3, lead induces phosphorylation of PERK and eIF2 in HAEC, indicating that the

The Keap1Nrf2 system is one of the major cellular defense mechanisms against oxidative and/or electrophilic stresses.56) Transcription factor Nrf2 recognizes the antioxidant response element/electrophile responsive element (ARE/EpRE) in the promoter region and regulates the basal and inducible expression of numerous antioxidant and detoxifying genes. Under normal conditions, Nrf2 is bound to Keap1, resulting in a lower level in the cells because of rapid proteasomal degradation. Keap1 acts as a negative regulator of Nrf2 and as a sensor of oxidative and/or electrophilic stress. Keap1 serves as a substrate linker protein for the interaction of the Cul3-based E3-ubiquitin ligase complex with Nrf2, leading to ubiquitination of Nrf2 and subsequent degradation by a proteasome. Once reactive thiols of Keap1 are modied by electrophiles or ROS, Cul3-dependent ubiquitination of Nrf2 is readily attenuated, thereby rescuing newly synthesized Nrf2 from proteasomal degradation and allowing for accumulation in the nucleus. The role of the reactive cysteine residues of Keap1 has

Fig. 3.

Lead Activates the PERK Pathway in HAEC

HAECs were exposed to 10 M Pb or 2 M thapsigargin (Tg) for the indicated times, and total cell lysates were subjected to Western blot analysis as described in Shinkai et al.33) with the indicated antibodies. Anti-PERK antibody and anti-phospho-PERK antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Anti-eIF2 antibody and phospho-eIF2 antibody were obtained from Cell Signaling Technology (Beverly, MA, U.S.A.).

November 2012

1889

Chart 1.

Cellular Defense Response to Lead via the UPR Pathway and the Keap1Nrf2 Pathway

been widely examined, and three residues, Cys151, Cys273 and Cys288, have been shown as critical to Keap1 function.56) Also, Dinkova-Kostova et al. reported that zinc is bound to reactive cysteine residues such as Cys273 and Cys288 and that this zinc can be replaced by electrophiles, leading to a conformational change in Keap1 and subsequent Nrf2 activation.57) If lead substitutes for zinc in the metal-binding site of Keap1, Nrf2 will be activated. In fact, Korashy et al. reported that lead can activate Nrf2 and upregulate NQO1 expression in Hepa1c1c7 cells.42) Recently, Zeller et al. demonstrated that lead activates Nrf2, thereby upregulating its downstream proteins in HUVEC.41) Consistent with these results, lead activates Nrf2 and its downstream proteins such as HO-1 and GCLM in HAEC (Fig. 2). Although the modication of cysteine residues of Keap1 appears to be a common mechanism for Nrf2 activation, the stimulation of phosphorylation signaling pathways may also underlie the ability to activate Nrf2 and its target genes.58) It has been reported that Nrf2 is phosphorylated by several kinases such as PKC and casein kinase 2, resulting in its activation. Cullinan et al. have identied Nrf2 as a direct PERK substrate and showed that ER stress triggers PERK-dependent Nrf2 nuclear accumulation.59) Thus, Nrf2 can be activated by lead via not only modication of a Keap1 thiol, but also through PKC and/or PERK-mediated Nrf2 phosphorylation. Once Nrf2 is activated, it transactivates a wide variety of ARE/EpRE-mediated enzymes. As shown in Table 1, lead induces Nrf2 downstream proteins such as HO-1, GCLM,

GSTA1, and NQO1. Because lead has been shown to have the capacity to bind thiol groups and to cause oxidative stress, these detoxifying and antioxidant enzymes would serve as part of a defense mechanism against lead toxicity in the vascular system.

7.

CONCLUDING REMARKS

The vascular system constitutes one of the key organ systems sustaining normal human physiology; however, its dysregulation also underlies multiple pathophysiologic processes. There is little doubt that lead exposure is one cause of vascular dysfunction through induction of cellular ER and/or oxidative stress. Cellular stress, mediated by lead, leads to an adaptive response involving various mechanisms including the UPR system and Keap1-Nrf2 system (Chart 1). In the case of endothelial cells, lead activates the IRE1-JNK-AP-1 pathway to up-regulate GRP78 and GRP94, thereby protecting cells from lead-induced cytotoxicity. Also, lead can activate Nrf2, presumably via modication of Keap1 and the PKC/PERK pathway, to increase antioxidant proteins such as HO-1 and GCLM in vascular endothelial cells. Because Nrf2 regulates not only antioxidant proteins but also phase II enzymes and phase III transporters, it will be a matter of future research to determine the role of Nrf2 in lead excretion into extracellular space. We believe that characterizing the cellular defense mechanisms against lead toxicity will provide helpful information for the development of intervention strategies to reduce

1890

Vol. 35, No. 11 18) Fujiwara Y, Kaji T, Yamamoto C, Sakamoto M, Kozuka H. Stimulatory effect of lead on the proliferation of cultured vascular smooth-muscle cells. Toxicology, 98, 105110 (1995). 19) Yamamoto C, Miyamoto A, Sakamoto M, Kaji T, Kozuka H. Lead perturbs the regulation of spontaneous release of tissue plasminogen activator and plasminogen activator inhibitor-1 from vascular smooth muscle cells and broblasts in culture. Toxicology, 117, 153161 (1997). 20) Yamamoto C, Kaji T. Effect of lead on the synthesis of tissue plasminogen activator by vascular endothelial cells in culture. J. Health Sci., 45, 119125 (1999). 21) Kursula P, Majava V. A structural insight into lead neurotoxicity and calmodulin activation by heavy metals. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun., 63, 653656 (2007). 22) Markovac J, Goldstein GW. Picomolar concentrations of lead stimulate brain protein kinase C. Nature, 334, 7173 (1988). 23) Sandhir R, Gill KD. Lead perturbs calmodulin dependent cyclic AMP metabolism in rat central nervous system. Biochem. Mol. Biol. Int., 33, 729742 (1994). 24) Hechtenberg S, Beyersmann D. Inhibition of sarcoplasmic reticulum Ca2+-ATPase activity by cadmium, lead and mercury. Enzyme, 45, 109115 (1991). 25) White LD, Cory-Slechta DA, Gilbert ME, Tiffany-Castiglioni E, Zawia NH, Virgolini M, Rossi-George A, Lasley SM, Qian YC, Basha MR. New and evolving concepts in the neurotoxicology of lead. Toxicol. Appl. Pharmacol., 225, 127 (2007). 26) Sharma SK, Goloubinoff P, Christen P. Heavy metal ions are potent inhibitors of protein folding. Biochem. Biophys. Res. Commun., 372, 341345 (2008). 27) Ahamed M, Siddiqui MK. Low level lead exposure and oxidative stress: current opinions. Clin. Chim. Acta, 383, 5764 (2007). 28) Inoguchi T, Sonta T, Tsubouchi H, Etoh T, Kakimoto M, Sonoda N, Sato N, Sekiguchi N, Kobayashi K, Sumimoto H, Utsumi H, Nawata H. Protein kinase C-dependent increase in reactive oxygen species (ROS) production in vascular tissues of diabetes: role of vascular NAD(P)H oxidase. J. Am. Soc. Nephrol., 14, S227S232 (2003). 29) Adonaylo VN, Oteiza PI. Pb2+ promotes lipid oxidation and alterations in membrane physical properties. Toxicology, 132, 1932 (1999). 30) Koch G, Smith M, Macer D, Webster P, Mortara R. Endoplasmic reticulum contains a common, abundant calcium-binding glycoprotein, endoplasmin. J. Cell Sci., 86, 217232 (1986). 31) Qian Y, Falahatpisheh MH, Zheng Y, Ramos KS, Tiffany-Castiglioni E. Induction of 78 kDa glucose-regulated protein (GRP78) expression and redox-regulated transcription factor activity by lead and mercury in C6 rat glioma cells. Neurotox. Res., 3, 581589 (2001). 32) Qian Y, Harris ED, Zheng Y, Tiffany-Castiglioni E. Lead targets GRP78, a molecular chaperone, in C6 rat glioma cells. Toxicol. Appl. Pharmacol., 163, 260266 (2000). 33) Shinkai Y, Yamamoto C, Kaji T. Lead induces the expression of endoplasmic reticulum chaperones GRP78 and GRP94 in vascular endothelial cells via the JNK-AP-1 pathway. Toxicol. Sci., 114, 378386 (2010). 34) Stacchiotti A, Morandini F, Bettoni F, Schena I, Lavazza A, Grigolato PG, Apostoli P, Rezzani R, Aleo MF. Stress proteins and oxidative damage in a renal derived cell line exposed to inorganic mercury and lead. Toxicology, 264, 215224 (2009). 35) Tchounwou PB, Yedjou CG, Foxx DN, Ishaque AB, Shen E. Lead-induced cytotoxicity and transcriptional activation of stress genes in human liver carcinoma (HepG2) cells. Mol. Cell. Biochem., 255, 161170 (2004). 36) Tully DB, Collins BJ, Overstreet JD, Smith CS, Dinse GE, Mumtaz MM, Chapin RE. Effects of arsenic, cadmium, chromium, and lead on gene expression regulated by a battery of 13 different promoters

the adverse effects of lead exposure. It has been reported that 4-phenylbutyric acid acts as a chemical chaperone to increase ER function.60) Also, we have previously reported that sulforaphane, an Nrf2 activator, has the ability to protect against arsenic and methylmercury toxicity by decreasing intracellular accumulation of the metal(loid).61,62) Therefore, treatment with 4-phenylbutyric acid and/or sulforaphane may be an effective strategy for the treatment of lead poisoning. Acknowledgments We thank Dr. Yasuyuki Fujiwara, Laboratory of Pharmaceutical Health Sciences, School of Pharmacy, Aichi Gakuin University, and Dr. Chika Yamamoto and Ms. Natsumi Iwasaki, Department of Environmental Health, Faculty of Pharmaceutical Sciences, Hokuriku University, for their contributions to the studies.

REFERENCES
1) Rosin A. The long-term consequences of exposure to lead. Isr. Med. Assoc. J., 11, 689694 (2009). 2) Vaziri ND. Mechanisms of lead-induced hypertension and cardiovascular disease. Am. J. Physiol. Heart Circ. Physiol., 295, H454 H465 (2008). 3) Verstraeten SV, Aimo L, Oteiza PI. Aluminium and lead: molecular mechanisms of brain toxicity. Arch. Toxicol., 82, 789802 (2008). 4) Patrick L. Lead toxicity, a review of the literature. Part 1: Exposure, evaluation, and treatment. Altern. Med. Rev., 11, 222 (2006). 5) Navas-Acien A, Guallar E, Silbergeld EK, Rothenberg SJ. Lead exposure and cardiovascular diseasea systematic review. Environ. Health Perspect., 115, 472482 (2007). 6) Menke A, Muntner P, Batuman V, Silbergeld EK, Guallar E. Blood lead below 0.48 mol/L (10 g/dL) and mortality among US adults. Circulation, 114, 13881394 (2006). 7) Navas-Acien A, Selvin E, Sharrett AR, Calderon-Aranda E, Silbergeld E, Guallar E. Lead, cadmium, smoking, and increased risk of peripheral arterial disease. Circulation, 109, 31963201 (2004). 8) Garza A, Vega R, Soto E. Cellular mechanisms of lead neurotoxicity. Med. Sci. Monit., 12, RA57RA65 (2006). 9) Patrick L. Lead toxicity part II: the role of free radical damage and the use of antioxidants in the pathology and treatment of lead toxicity. Altern. Med. Rev., 11, 114127 (2006). 10) Qian Y, Tiffany-Castiglioni E. Lead-induced endoplasmic reticulum (ER) stress responses in the nervous system. Neurochem. Res., 28, 153162 (2003). 11) Prozialeck WC, Edwards JR, Nebert DW, Woods JM, Barchowsky A, Atchison WD. The vascular system as a target of metal toxicity. Toxicol. Sci., 102, 207218 (2008). 12) Kaji T. Cell biology of heavy metal toxicity in vascular tissue. Yakugaku Zasshi, 124, 113120 (2004). 13) Fujiwara Y, Kaji T, Sakurai S, Sakamoto M, Kozuka H. Inhibitory effect of lead on the repair of wounded monolayers of cultured vascular endothelial cells. Toxicology, 117, 193198 (1997). 14) Fujiwara Y, Watanabe S, Sakamoto M, Kaji T. Repair of wounded monolayers of cultured vascular endothelial cells after simultaneous exposure to lead and zinc. Toxicol. Lett., 94, 181188 (1998). 15) Kaji T, Fujiwara Y, Hoshino M, Yamamoto C, Sakamoto M, Kozuka H. Inhibitory effect of lead on the proliferation of cultured vascular endothelial cells. Toxicology, 95, 8792 (1995). 16) Kaji T, Ohkawara S, Nakajima M, Yamamoto C, Fujiwara Y, Miyajima S, Koizumi F. Lead-induced alteration of heparan sulfate proteoglycans in cultured vascular endothelial cells. Toxicology, 118, 110 (1997). 17) Fujiwara Y, Kaji T. Lead inhibits the core protein synthesis of a large heparan sulfate proteoglycan perlecan by proliferating vascular endothelial cells in culture. Toxicology, 133, 159169 (1999).

November 2012 in recombinant HepG2 cells. Toxicol. Appl. Pharmacol., 168, 7990 (2000). Qian Y, Zheng Y, Ramos KS, Tiffany-Castiglioni E. GRP78 compartmentalized redistribution in Pb-treated glia: role of GRP78 in lead-induced oxidative stress. Neurotoxicology, 26, 267275 (2005). Cabell L, Ferguson C, Luginbill D, Kern M, Weingart A, Audesirk G. Differential induction of heme oxygenase and other stress proteins in cultured hippocampal astrocytes and neurons by inorganic lead. Toxicol. Appl. Pharmacol., 198, 4960 (2004). Korashy HM, El-Kadi AO. The role of redox-sensitive transcription factors NF-kappaB and AP-1 in the modulation of the Cyp1a1 gene by mercury, lead, and copper. Free Radic. Biol. Med., 44, 795806 (2008). Vargas H, Castillo C, Posadas F, Escalante B. Acute lead exposure induces renal haeme oxygenase-1 and decreases urinary Na+ excretion. Hum. Exp. Toxicol., 22, 237244 (2003). Zeller I, Knoach M, Seubert A, Kreutmayer SB, Stelzmller ME, Wallnoefer E, Blunder S, Frotschnig S, Messner B, Willeit J, Debbage P, Wick G, Kiechl S, Laufer G, Bernhard D. Lead contributes to arterial intimal hyperplasia through nuclear factor erythroid 2-related factor-mediated endothelial interleukin 8 synthesis and subsequent invasion of smooth muscle cells. Arterioscler. Thromb. Vasc. Biol., 30, 17331740 (2010). Korashy HM, El-Kadi AO. Transcriptional regulation of the NAD(P)H:quinone oxidoreductase 1 and glutathione S -transferase Ya genes by mercury, lead, and copper. Drug Metab. Dispos., 34, 152165 (2006). Korashy HM, El-Kadi AO. NF-kappaB and AP-1 are key signaling pathways in the modulation of NAD(P)H:quinone oxidoreductase 1 gene by mercury, lead, and copper. J. Biochem. Mol. Toxicol., 22, 274283 (2008). Scortegagna M, Chikhale E, Hanbauer I. Lead exposures increases oxidative stress in serum deprived E14 mesencephalic cultures. Role of metallothionein and glutathione. Restor. Neurol. Neurosci., 12, 95101 (1998). Yu J, Fujishiro H, Miyataka H, Oyama TM, Hasegawa T, Seko Y, Miura N, Himeno S. Dichotomous effects of lead acetate on the expression of metallothionein in the liver and kidney of mice. Biol. Pharm. Bull., 32, 10371042 (2009). Chou YH, Woon PY, Huang WC, Shiurba R, Tsai YT, Wang YS, Hsieh TJ, Chang WC, Chuang HY, Chang WC. Divalent lead cations induce cyclooxygenase-2 gene expression by epidermal growth factor receptor/nuclear factor-kappa B signaling in A431carcinoma cells. Toxicol. Lett., 203, 147153 (2011). Courtois E, Marques M, Barrientos A, Casado S, Lpez-Farr A. Lead-induced downregulation of soluble guanylate cyclase in isolated rat aortic segments mediated by reactive oxygen species and cyclooxygenase-2. J. Am. Soc. Nephrol., 14, 14641470 (2003).

1891 48) Xu J, Ji LD, Xu LH. Lead-induced apoptosis in PC 12 cells: involvement of p53, Bcl-2 family and caspase-3. Toxicol. Lett., 166, 160167 (2006). 49) Xu J, Lian LJ, Wu C, Wang XF, Fu WY, Xu LH. Lead induces oxidative stress, DNA damage and alteration of p53, Bax and Bcl-2 expressions in mice. Food Chem. Toxicol., 46, 14881494 (2008). 50) Struzyska L, Bubko I, Walski M, Rafaowska U. Astroglial reaction during the early phase of acute lead toxicity in the adult rat brain. Toxicology, 165, 121131 (2001). 51) Bandyopadhyay U, Sridhar S, Kaushik S, Kif n R, Cuervo AM. Identi cation of regulators of chaperone-mediated autophagy. Mol. Cell, 39, 535547 (2010). 52) Bouton CM, Hossain MA, Frelin LP, Laterra J, Pevsner J. Microarray analysis of differential gene expression in lead-exposed astrocytes. Toxicol. Appl. Pharmacol., 176, 3453 (2001). 53) Pan TL, Wang PW, Al-Suwayeh SA, Chen CC, Fang JY. Skin toxicology of lead species evaluated by their permeability and proteomic proles: a comparison of organic and inorganic lead. Toxicol. Lett., 197, 1928 (2010). 54) Ron D, Walter P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat. Rev. Mol. Cell Biol., 8, 519529 (2007). 55) Walter P, Ron D. The unfolded protein response: from stress pathway to homeostatic regulation. Science, 334, 10811086 (2011). 56) Taguchi K, Motohashi H, Yamamoto M. Molecular mechanisms of the Keap1Nrf2 pathway in stress response and cancer evolution. Genes Cells, 16, 123140 (2011). 57) Dinkova-Kostova AT, Holtzclaw WD, Wakabayashi N. Keap1, the sensor for electrophiles and oxidants that regulates the phase 2 response, is a zinc metalloprotein. Biochemistry, 44, 68896899 (2005). 58) Copple IM, Goldring CE, Kitteringham NR, Park BK. The Nrf2-Keap1 defence pathway: role in protection against drug-induced toxicity. Toxicology, 246, 2433 (2008). 59) Cullinan SB, Zhang D, Hannink M, Arvisais E, Kaufman RJ, Diehl JA. Nrf2 is a direct PERK substrate and effector of PERK-dependent cell survival. Mol. Cell. Biol., 23, 71987209 (2003). 60) Papp E., Csermely P. Chemical chaperones: mechanisms of action and potential use. Handb. Exp. Pharmacol., 2006, 405416 (2006). 61) Shinkai Y, Sumi D, Fukami I, Ishii T, Kumagai Y. Sulforaphane, an activator of Nrf2, suppresses cellular accumulation of arsenic and its cytotoxicity in primary mouse hepatocytes. FEBS Lett., 580, 17711774 (2006). 62) Toyama T, Shinkai Y, Yasutake A, Uchida K, Yamamoto M, Kumagai Y. Isothiocyanates reduce mercury accumulation via an Nrf2-dependent mechanism during exposure of mice to methylmercury. Environ. Health Perspect., 119, 11171122 (2011).

37)

38)

39)

40)

41)

42)

43)

44)

45)

46)

47)