山莨菪碱对大鼠胸水中性白细胞花生四烯酸代谢的影响

本文研究了654-2对大鼠胸水中性白细胞代谢[1-¹⁴C]AA及内源性AA的影响。大鼠白细胞AA经5-LPO代谢途径形成的主要产物为LTB₄及5-HETE,经CO途径的主要产物为HHT及TXB₂。654-2对白细胞代谢[1-¹⁴C]AA无抑制作用,但显著减少白细胞从内源性AA形成的LTB₄,5-HETE,HHT及TXB₂。这种抑制作用与654-2呈剂量依赖关系。本实验结果表明,654-2抑制PG及LT的形成可能是影响了AA从胞膜的释放,而并非直接抑制CO及5-LPO。     

The anti-tumourigenic effect of ellagic acid in SKOV-3 ovarian cancer cells entails activation of autophagy mediated by inhibiting Akt and activating AMPK

This study investigated the effect of ellagic acid (EA) on SKOV-3 cell growth and invasiveness and tested if the underlying mechanism involves modulating autophagy. Cells were treated with EA in the presence or absence of chloroquine (CQ), an autophagy inhibitor, compound C (CC), an AMPK inhibitor, or an insulin-like growth factor-1 (IGF-1), a PI3K/Akt activator. EA, at an IC₅₀ of 36.6 µmol/L, inhibited cell proliferation, migration, and invasion and induced cell apoptosis in SKOV-3 cells. These events were prevented by CQ. Also, EA increased levels of Beclin-1, ATG-5, LC3I/II, Bax, cleaved caspase-3/8 and reduced those of p62 and Bcl-2 in these cancer cells. Mechanistically, EA decreased levels of p-S6K1 (Thr³⁸⁹) and 4EBP-1 (Thr^37/46), two downstream targets of mTORC1, and p-Akt (Thr³⁰⁸) but increased levels of AMPK (Thr¹⁷²) and p-raptor (Ser⁷⁹²), a natural inhibitor of mTORC1. CC or IGF-1 alone partially prevented the effect of EA on cell survival, cell invasions, and levels of LDH, Beclin-1, and cleaved caspase-3. In conclusion, EA can inhibit SKOV-3 growth, migration, and invasion by activating cytotoxic autophagy mediated by inhibition of mTORC1 and Akt and activation of AMPK.     

Growth inhibitory effect of KYKZL-1 on Hep G2 cells via inhibition of AA metabolites and caspase-3 pathway and cell cycle arrest

KYKZL-1, a newly synthesized compound with COX/5-LOX dual inhibition, was subjected to the inhibitory activity test on Hep G₂ growth. We found that KYKZL-1 inhibited the growth of Hep G₂ cells via inducing apoptosis. Further studies showed that KYKZL-1 activated caspase-3 through cytochrome c release from mitochondria and down regulation of Bcl-2/Bax ratio and reduced the high level of COX-2 and 5-LOX. As shown in its anti-inflammatory effect, KYKZL-1 also exhibited inhibitory effect on the PGE₂ and LTB₄ production in Hep G₂ cells. Accordingly, exogenous addition of PGE₂ or LTB₄ reversed the decreases in cell viability. In addition, KYKZL-1 caused cell cycle arrest at the S–G₂ checkpoint via the activation of p21^CIP1 protein and down-regulation of cyclin A expression.These data indicate that the growth inhibitory effect of KYKZL-1 is associated with inhibition of AA metabolites and caspase-3 pathway and cell cycle arrest.Combined with our previous findings, KYKZL-1 exhibiting COX/5-LOX inhibition may be a promising potential agent not only for inflammation control but also for cancer prevention/therapy with an enhanced gastric safety profile.     

Induction of leukotriene B4 and prostaglandin E2 release from keratinocytes by protease-activated receptor-2-activating peptide in ICR mice

Protease-activated receptor-2 (PAR2) has been shown to play a key role in the pathophysiology of itch. However, the precise mechanism of PAR2-mediated itch remains largely unknown. In the present study, we investigated the effects of several agents on the scratching behavior induced by PAR2-activating peptide (SLIGRL-NH₂). Pretreatment of experimental animals with tacrolimus or the 5-lipoxygenase inhibitor zileuton significantly reduced SLIGRL-NH₂-induced scratching behavior, whereas histamine H₁ receptor antagonist cetirizine or the cyclooxygenase inhibitor indomethacin had little effect. Furthermore, intradermal injection of SLIGRL-NH₂ increased cutaneous levels of LTB₄ and PGE₂. In vitro, SLIGRL-NH₂ treatment enhanced LTB₄ and PGE₂ release from primary keratinocytes in a concentration-dependent manner. Preincubation of keratinocytes with zileuton resulted in a significant decrease of LTB₄ release and treatment of indomethacin led to a significant decrease of PGE₂ in response to SLIGRL-NH₂ stimulation. In addition, SLIGRL-NH₂-induced secretion of LTB₄ and PGE₂ was significantly inhibited by tacrolimus, whereas cetirizine had no effect. These results indicate that SLIGRL-NH₂ stimulates LTB₄ and PGE₂ release from mouse keratinocytes and that enhancement of LTB₄ and PGE₂ secretion contributes to SLIGRL-NH₂-induced scratching behavior in ICR mice.     

Cadmium exposure activates Akt/ERK Signaling and pro-inflammatory COX-2 expression in human gallbladder epithelial cells via a ROS dependent mechanism

Gallbladder cancer (GBC) is the commonest biliary tract cancer with an ill-defined etiology. We examined the role of Cd⁺² exposures in a primary human gallbladder (GB) cell line model in this study. Cd⁺² exposures induced decreased cell viability, reactive oxygen species (ROS) generation, altered Akt/ERK signaling pathway activation, PGE₂ and COX-2 expression in a human primary gallbladder epithelial cell model. Pharmacological inhibitors were used to determine the key drivers of elevated COX-2 expression due to Cd⁺² exposure. Our results show Cd⁺² causes a dose-dependent reduction in GB cell viability (EC50 value – 18.6 μM). Dose-dependent activation of phospho-Akt and phospho-ERK signaling pathways via increased phosphoprotein expression was observed due to Cd⁺². Signaling activation of Akt and ERK was prevented by 5 mM N-Acetyl Cysteine (NAC), establishing the role of ROS as a key driver in the activation process. Importantly, we observed Cd⁺² also caused a dose dependent change in the COX-2 and PGE₂ expression levels. PI3K-Akt and NF-kB signaling pathways play a key role in Cd⁺² exposure induced COX-2 activation in the gallbladder epithelial cells. In conclusion, our study measures the toxicological effects of Cd⁺² exposures on human GB epithelial cells for the first time and establishes the role of Cd⁺² as a possible driver of the Akt/ERK pathway overactivity and chronic inflammation in gallbladder carcinogenesis.     

Angiotensin-induced EGF receptor transactivation inhibits insulin signaling in C9 hepatic cells

To investigate the potential interactions between the angiotensin II (Ang II) and insulin signaling systems, regulation of IRS-1 phosphorylation and insulin-induced Akt activation by Ang II were examined in clone 9 (C9) hepatocytes. In these cells, Ang II specifically inhibited activation of insulin-induced Akt Thr³⁰⁸ and its immediate downstream substrate GSK-3α/β in a time-dependent fashion, with ∼70% reduction at 15 min. These inhibitory actions were associated with increased IRS-1 phosphorylation of Ser⁶³⁶/Ser⁶³⁹ that was prevented by selective blockade of EGFR tyrosine kinase activity with AG1478. Previous studies have shown that insulin-induced phosphorylation of IRS-1 on Ser⁶³⁶/Ser⁶³⁹ is mediated mainly by the PI3K/mTOR/S6K-1 sequence. Studies with specific inhibitors of PI3K (wortmannin) and mTOR (rapamycin) revealed that Ang II stimulates IRS-1 phosphorylation of Ser⁶³⁶/Ser⁶³⁹ via the PI3K/mTOR/S6K-1 pathway. Both inhibitors blocked the effect of Ang II on insulin-induced activation of Akt. Studies using the specific MEK inhibitor, PD98059, revealed that ERK1/2 activation also mediates Ang II-induced S6K-1 and IRS-1 phosphorylation, and the impairment of Akt Thr³⁰⁸ and GSK-3α/β phosphorylation. Further studies with selective inhibitors showed that PI3K activation was upstream of ERK, suggesting a new mechanism for Ang II-induced impairment of insulin signaling. These findings indicate that Ang II has a significant role in the development of insulin resistance by a mechanism that involves EGFR transactivation and the PI3K/ERK1/2/mTOR-S6K-1 pathway.     

857-860Anti-inflammatory Effect of Magnolol,Isolated from Magnolia officinalis,on A23187-induced Pleurisy in Mice

In the present study, A23187-induced pleurisy in mice was used to investigate the anti-inflammatory effect of magnolol, a phenolic compound isolated from Chinese medicine Hou p'u (cortex of Magnolia officinalis). A23187-induced protein leakage was reduced by magnolol (10mgkg^?1, i.p.), indomethacin (10mgkg^?1, i.p.) and BW755C (30mgkg^?1, i.p.). A23187-induced polymorphonuclear (PMN) leucocyte infiltration in the pleural cavity was suppressed by magnolol and BW755C, while enhanced by indomethacin. Like BW755C, magnolol reduced both prostaglandin E₂ (PGE₂) and leukotriene B₄ (LTB₄) levels in the pleural fluid of A23187-induced pleurisy, while indomethacin reduced PGE₂ but increased LTB₄ formation. In the rat isolated peripheral neutrophil suspension, magnolol (3.7 μM) and BW755C (10 μM) also suppressed the A23187-induced thromboxane B₂ (TXB₂) and LTB₄ formation. These results suggest that magnolol, like BW755C, might be a dual cyclo-oxygenase and lipoxygenase inhibitor. The inhibitory effect of magnolol on the A23187-induced pleurisy is proposed to be, at least partly, dependent on the reduction of the formation of eicosanoids mediators in the inflammatory site.     

The antiinflammatory action of guanabenz is mediated through 5-lipoxygenase and cyclooxygenase inhibition

Guanabenz (2,6-dichlorobenzylidene amino guanidine acetate), an α₂-agonist, possesses antiinflammatory substances, the effect of guanabenz on LT and Since leukotrienes (LT) and prostaglandins(PG) are proinflammatory substances, the effect of guanabenz on LT and PG synthesis by inflammatory cells was investigated. Guanabenz, but not clonidine, B-HT 920 or B-HT 933 inhibited zymosan-induced LTC₄ (IC₅₀=13 μM) and PGE₂ (IC₅₀=10.9 μM) synthesis with no concomitant reduction in zymosan phagocytosis or cell viability. Similarlyguanabenz reduced LTB₄ (IC₅₀=37.4 μM) and PGE₂ (IC₅₀=13.8 μM) synthesis by A23187-stimulated rat glycogen elicited neutrophils. Furthermore, guanabenz did not inhibit platelet 12-lipoxygenase or phospholipase A₂. In vivo, guanabenz was orally active against rat carrageenan paw edema and adjuvant arthritis (ED_50^s=9 and 10 mg/kg, respectively). Topically applied guanabenz reduced arachidonic acid (AA)- or tetradecanoyl phorbol acetate (TPA)-induced ear inflammation (ED_50^s:AAA-induced ear edema, 1.4 mg/ear; PMA-induced ear edema, 0.013 mg/ear). Therefore, the antiinflammatory activity of guanabenz may be due to its ability to inhibit the formation of 5-lipoxygenase and cyclooxygenase products.     

Biphasic effect of cadmium ions on the secretion of leukotriene B4 in rabbit alveolar macrophages

 One major role of alveolar macrophages is the production of eicosanoids, which modulate immune and inflammatory processes in the lung. In this study, the effects were investigated of cadmium ions on the secretion of leukotriene (LT)B₄ and prostaglandin (PG)E₂, predominant products of lipoxygenase and cyclooxygenase, respectively. Cd²⁺ had an inhibitory effect on the secretion of LTB₄ and PGE₂ in response to A23187 stimulation at concentrations >3× 10^-5 M. This effect can be explained by the inhibition of arachidonic acid (20 : 4) liberation from membrane phospholipids by Cd²⁺, because Cd²⁺ inhibits both [³H]arachidonic acid (20 : 4) liberation from [³H]20 : 4-prelabeled macrophages and the cytosolic phospholipase A₂ activity. At concentrations <3×10^-5 M, Cd²⁺ had no effect on PGE₂ secretion but showed an augmentation of LTB₄ secretion. In vitro study using macrophage lysate showed enhanced LTB₄ synthesis from arachidonic acid by Cd²⁺, which could be responsible for the augmentation of LTB₄ secretion in cells. These results indicate that Cd²⁺ may increase inflammation by increasing LTB₄ production in lung. Received: 13 February 1996/Accepted: 23 April 1996     

Inhibition of Leukotriene Biosynthesis and Polymorphonuclear Leukocyte Functions by Orally Active Quinolylmethoxyphenylamines

Abstract: The N-substituted quinolylmethoxyphenylamines, ETH603, ETH615 and ETH647, inhibited the formation of LTB₄ in rat peritoneal leukocytes, human peripheral polymorphonuclear leukocytes and canine whole blood. In rat and human cells, the compounds also inhibited the formation of 5-HETE and stimulated the synthesis of 15-HETE. In rat leukocytes, the compounds were 15–30 times more potent inhibitors of LTB₄ synthesis than nordihydroguaiaretic acid, but in canine whole blood they were significantly less potent, possibly due to protein binding. However, after oral administration of the compounds to dogs a long-lasting inhibition of LTB₄ production in peripheral blood was observed at serum concentrations much lower than those required in vitro. Furthermore, the compounds inhibited the LTB₄-directed chemotaxis and the phagocytosis of C. albicans blastospores by canine polymorphonuclear leukocytes both in vitro and following oral administration. The calcium ionophore A23187-induced release of LTB₄ in the peritoneal cavity of rats was also inhibited by systemic administration of the compounds. We therefore conclude that these novel quinolines are orally active 5-lipoxygenase inhibitors which may accumulate in inflammatory cells in vivo, leading to potent inhibition of leukotriene biosynthesis and cell function.     

Effect of norathyriol,isolated from Tripterospermum lanceolatum,on A 23 187-induced pleurisy and analgesia in mice

A 23 187-induced pleurisy in the mouse was demonstrated in this study. The protein leakage, leukocyte accumulation, LTB₄ and PGE₂ production in the pleural cavity of mice were increased by A 23 187 in a dose-dependent manner. At 7.5 nmole A 23 187 intrapleural injection, the protein level peaked at 0.5–2 h, PMN leukocytes accumulation peaked at 3–4 h, and LTB₄ and PGE₂ production peaked at 0.5–1 h. In this in vivo model we investigated the anti-inflammatory effect of norathyriol, isolated from Tripterospermum lanceolatum. A 23 187-induced protein leakage was reduced by norathyriol (ID₅₀ was about 30.6 mg/kg i.p.), indomethacin and BW 755 C. A 23 187-induced PMN leukocytes accumulation was suppressed by norathyriol (ID₅₀ was about 16.8 mg/kg, i.p.) and BW 755 C, while enhanced by indomethacin. Like BW 755 C, norathyriol reduced both LTB₄ and PGE₂ production (ID₅₀ was about 18.6and 29.1 mg/kg i.p., respectively), while indomethacin reduced PGE₂ but not LTB₄ generation. We also demonstrated the analgesic effect of norathyriol on the acetic acid-induced writhing response. Acetic acid-induced writhing response was depressed by norathyriol (ID₅₀ was about 27.9 mg/kg i.p.), indomethacin and ibuprofen. These results suggest that norathyriol, like BW 755 C, might be a dual, yet weak, cyclooxygenase and lipoxygenase pathway blocker. The inhibitory effect of norathyriol on the A 23 187-induced pleurisy and acetic acid-induced writhing response in mice is proposed to be dependent on the reduction of eicosanoids mediators formation in the inflammatory site. Correspondence to: J.-P. Wang at the above address     

The development of methodology for clinical measurement of 5-lipoxygenase pathway intermediates from human peripheral blood mononuclear cells

Recent studies have shown a correlation between 5-lipoxygenase (5-LO) pathway up-regulation and cardiovascular risk. Despite the existence of several assays for products of the 5-LO pathway, a reliable method for clinical determination of 5-LO activity remains to be established. In the present communication, we report conditions that allow measurement of 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B₄ (LTB₄) in peripheral blood mononuclear cells (PBMCs) isolated from the blood of atherosclerosis patients before and after stimulation by the calcium ionophore, A23187. LTB₄, a potent mediator of inflammation-linked cardiovascular disease, was measured using an existing competitive enzyme immunoassay (EIA) kit after making specific methodological improvements that allowed PBMCs to be used in this format for the first time. LTB₄ was also measured by LC/MS/MS along with 5-HETE, a direct by-product of the action of 5-LO on arachidonic acid and a molecule for which no commercial EIA kit exists. The LC/MS/MS assay was validated over a range of 0.025–25 ng/mL for LTB₄ and 0.1–25 ng/mL for 5-HETE. The EIA method has a validated range covering 0.025–4 ng/mL. When both assays were applied to analyze LTB₄ from stimulated PBMCs isolated from 25 subjects with various degrees of atherosclerosis, a high correlation was obtained (r = 0.9426, Pearson's correlation coefficient). A high correlation was also observed between the levels of LTB₄ and 5-HETE measured by LC/MS/MS after ionophore stimulation (r = 0.9159). Details are presented for optimized sample collection, processing, storage, and analysis in accordance with the logistical demands of clinical analysis.     

The generation and metabolism of leukotrienes in the ionophore-stimulated blood of normal and asthmatic subjects

The generation and metabolism of leukotrienes (LTs) 1341 C₄, D4, and E₄ were studied in vitro in the A23187-stimulated whole blood of normal (N) and atopic asthmatic (AA) human subjects. Using a combination of reversed-phase high performance liquid chromatography and radioimmunoassay, we have demonstrated that the blood cells of atopic asthmatic patients have an enhanced ability to release LTB4 and LTC4 when compared to those of normal subjects. The release of LTB₄ and LTC₄ in response to ionophore is dose- and time-dependent. Half-maximal doses of ionophore caused the generation of high, sustained levels of LTB₄, which are significantly higher in the AA blood than in N blood. Incubations of ³H-LTB₄ in ionophore-stimulated N and AA blood revealed a slow metabolism to 20-OH-LTB₄ and 20-COON-LTB₄.LTC₄ is generated in smaller amounts than LTB4, with an early peak after 10 min which is significantly higher (p < 0.01) in the AA blood compared to the N blood. Subsequent metabolism of LTC₄ elicits significantly greater amounts of LTD₄, and consistently higher levels of LTE₄, in the AA blood. Parallel incubations of 3H-LTC₄ in ionophore-stimulated N and AA blood demonstrated rapid metabolism of LTC4 by the glutathione detoxification pathway. The elevated production of LTB₄ and LTC₄ in AA blood was not accounted for by differences in leukocyte sub-type counts in the two groups, nor by differences in their rates of catabolism. The novel, selective 5-lipoxygenase inhibitor BW A4C [N-(3-phenoxycinnamyl) acetohydroxamic acid] caused dose-dependent inhibition of LTB₄ and LTC₄ generation and was equipotent in N and AA blood.     

Tamoxifen inhibits 5-lipoxygenase in human polymorphonuclear leucocytes

Breast cancer patient survival is increased by tamoxifen, and we therefore need to understand how this drug exerts its effect. We describe a novel action of tamoxifen, the inhibition of LTB₄ and 5-HETE production from [¹⁴C]-arachidonic acid by human polymorphonuclear leucocytes.     

Antitumor and immunostimulatory activity of a polysaccharide–protein complex from Scolopendra subspinipes mutilans L. Koch in tumor-bearing mice

Scolopendra subspinipes mutilans L. Koch has been used for cancer treatment in traditional Chinese medicine for hundreds of years. In this study, the effects of a polysaccharide–protein complex from Scolopendra subspinipes mutilans L. Koch (SPPC) on the tumor growth and immune function were assessed in sarcoma S180 and hepatoma H22 bearing mice. Results showed that SPPC significantly inhibited the growth of S180 transplanted in mice and prolonged the survival time of H22- bearing mice. In S180-bearing mice, it promoted specific and nonspecific immune response as evidenced by enhancing the activities of natural killer (NK) cells, cytotoxic T lymphocytes (CTL) and the ratio of Th1/Th2 cytokines, and increasing the percentages of CD4⁺ T cells, B cells and NK cells. Furthermore, SPPC not only significantly inhibited mRNA expression and production of the immunosuppressive cytokines (IL-10 and TGF-β), but also diminished arachidonic acid (AA)-metabolizing enzymes (COX-2 and CYP4A) and their products (PGE₂ and 20-HETE) in tumor-associated macrophages (TAMs). Taken together, our results indicate that SPPC inhibits tumor growth in vivo by improving antitumor immune responses at least partly via downregulating AA-metabolic pathways in TAMs, and could act as an anti-tumor agent with immunomodulatory activity.     

Effect of Nedocromil Sodium on Sulfidopeptide Leukotrienes-stimulated Human Alveolar Macrophages in Asthma

Summary: Alveolar macrophages (AM) may take part in the amplification of the inflammatory mechanism involved in asthma. During an asthma attack, mast cells and eosinophils release arachidonic acid derivative mediators of inflammation such as sulfidopeptide leukotrienes. Among them, LTC₄ has been shown to be present in bronchoalveolar fluid. In asthmatic patients, we showed that the ability of AM to transform LTC₄ into its derivatives LTD₄ and LTE₄ was related to the intensity of the local inflammation observed during endoscopy. AM from asthmatics incubated in the presence of LTC₄ or LTE₄, generated LTB₄ and 5-HETE, which are potent chemoattractants. Nedocromil sodium (10^-4 M) decreased LTB₄ releasability and intracellular 5-HETE concentrations in zymosan-stimulated AM from asthmatic patients, and was shown to decrease the LTC₄ or LTE₄-promoted formation of LTB₄ and 5-HETE.     

Psoralidin, a dual inhibitor of COX-2 and 5-LOX, regulates ionizing radiation (IR)-induced pulmonary inflammation

Radiotherapy is the most significant non-surgical cure for the elimination of tumor, however it is restricted by two major problems: radioresistance and normal tissue damage. Efficiency improvement on radiotherapy is demanded to achieve cancer treatment. We focused on radiation-induced normal cell damage, and are concerned about inflammation reported to act as a main limiting factor in the radiotherapy. Psoralidin, a coumestan derivative isolated from the seed of Psoralea corylifolia, has been studied for anti-cancer and anti-bacterial properties. However, little is known regarding its effects on IR-induced pulmonary inflammation. The aim of this study is to investigate mechanisms of IR-induced inflammation and to examine therapeutic mechanisms of psoralidin in human normal lung fibroblasts and mice. Here, we demonstrated that IR-induced ROS activated cyclooxygenases-2 (COX-2) and 5-lipoxygenase (5-LOX) pathway in HFL-1 and MRC-5 cells. Psoralidin inhibited the IR-induced COX-2 expression and PGE₂ production through regulation of PI3K/Akt and NF-κB pathway. Also, psoralidin blocked IR-induced LTB₄ production, and it was due to direct interaction of psoralidin and 5-lipoxygenase activating protein (FLAP) in 5-LOX pathway. IR-induced fibroblast migration was notably attenuated in the presence of psoralidin. Moreover, in vivo results from mouse lung indicate that psoralidin suppresses IR-induced expression of pro-inflammatory cytokines (TNF-α, TGF-β, IL-6 and IL-1 α/β) and ICAM-1. Taken together, our findings reveal a regulatory mechanism of IR-induced pulmonary inflammation in human normal lung fibroblast and mice, and suggest that psoralidin may be useful as a potential lead compound for development of a better radiopreventive agent against radiation-induced normal tissue injury.     

Detection of key enzymes,free radical reaction products and activated signaling molecules as biomarkers of cell damage induced by benzo[a]pyrene in human keratinocytes

Benzo[a]pyrene (BaP) is a known carcinogenic and cell damaging agent. The underlying cell damaging pathomechanisms have not been totally revealed. Especially BaP-related induction of oxidative and nitrosative stress has not been previously investigated in detail. The presented study investigated these effects in order to elucidate the pathomechanism and as well to identify potential biological markers that may indicate a BaP exposure. Human immortalized keratinocytes (HaCaT cells) were exposed to BaP (1 μM) for either 5 min or 6 h, respectively. BaP-induced cellular damage was evaluated by immunocytochemistry analysis of multiple signaling cascades (e.g. apoptosis, Akt, MAPK, NOS, nitrotyrosine and 8-isoprostane formation), detection of nitrosative stress using diaminofluorescein (DAF-FM) and oxidative stress using 3′ -(p-aminophenyl)fluorescein (APF).Our results show that BaP exposure significantly enhanced NO and ROS productions in HaCaT cells. BaP led to eNOS-phosphorylation at Ser¹¹⁷⁷, Thr⁴⁹⁵ and Ser¹¹⁶ residues. Using specific inhibitors, we found that the Erk1/2 pathways seemed to have strong impact on eNOS phosphorylation. In addition, BaP-induced apoptosis was observed by caspase-3 activation and PARP cleavage.Our results suggest that BaP mediates its toxic effect in keratinocytes through oxidative and nitrosative stress which is accompanied by complex changes of eNOS phosphorylation and changes of Akt and MAPK pathways.