Phthalate plasticizer di(2‐ethyl‐hexyl) phthalate induces cyclooxygenase‐2 expression in gastric adenocarcinoma cells

The phthalate plasticizer, di(2‐ethyl‐hexyl) phthalate (DEHP), and its derived metabolites are common anthropogenic environmental toxins, which are known to act as endocrine disruptors. Numerous studies have associated DEHP with disruption of sex hormones, abnormal development of reproductive organs, allergies, and inflammation. Its role in promoting inflammation has been reported by both human epidemiological and animal studies. In stomach tissue, chronic inflammation is known to accompany mucosal damage, and pave the way to gastritis, stomach ulcers, and ultimately gastric cancer. Eastern Asian populations possess the highest gastric cancer incidences in the world. Coincidentally, East Asia is one of the world's major sites for plastics manufacture and export. Thus, possible correlations between DEHP, a common plasticizer, and gastric cancer are of great interest. Our study revealed several critical findings. First, even at very low dosage, mimicking the residual plasticizer exposure, detrimental effects of DEHP on gastric cells can be detected. Second, gastric cells treated with DEHP increased cyclooxygenase‐2 (COX‐2) in a time‐dependent manner. Third, promoter deletion studies revealed a critical role of nuclear factor‐kappa B (NF‐κB) for COX‐2 gene responses. Finally, our results indicated that a low concentration of DEHP is able to trigger COX‐2 activation via the extracellular signal–regulated kinase (ERK1/2) and NF‐κB signaling pathway. Taken together, we demonstrate that very low doses of DEHP enhance the expression of the prototypical inflammatory gene, COX‐2, in gastric cancer cells via ERK1/2 and NF‐κB activation. This study provides important insights into the inflammatory process and damages associated with phthalate plasticizers exposure.     

Involvement of L‐type Ca2+ channel and toll‐like receptor‐4 in nickel‐induced interleukin‐8 gene expression

The metal nickel (Ni²⁺) is found everywhere in our daily lives, including coins, costume jewelry, and even nuts and chocolates. Nickel poisoning can cause inflammatory reactions, respiratory diseases, and allergic contact dermatitis. To clarify the mechanism by which nickel induces mediators of inflammation, we used the human acute monocytic leukemia THP‐1 cell line as a model. Interleukin (IL)‐8 promoter activity as well as gene expression were tested by luciferase assay and real‐time polymerase chain reaction. The underlying mechanisms of nickel‐induced IL‐8 were investigated. We found that nickel induced IL‐8 gene expression via the L‐type Ca²⁺ channel, Toll‐like receptor‐4 (TRL‐4) and nuclear factor NF‐κB signal transduction pathways. Nickel activated NF‐κB expression through extracellular signal‐regulated kinase 1/2 phosphorylation and then increased IL‐8 expression. Thus, the L‐type Ca²⁺ channel and TRL‐4 play important roles in nickel‐induced inflammatory gene expressions. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 5–12, 2016.     

Synthesis of deuterium‐labeled analogs of the lipid hydroperoxide‐derived bifunctional electrophile 4‐oxo‐2(E)‐nonenal

Lipid hydroperoxides undergo homolytic decomposition into the bifunctional 4‐hydroxy‐2(E)‐nonenal and 4‐oxo‐2(E)‐nonenal (ONE). These bifunctional electrophiles are highly reactive and can readily modify intracellular molecules including glutathione (GSH), deoxyribonucleic acid (DNA) and proteins. Lipid hydroperoxide‐derived bifunctional electrophiles are thought to contribute to the pathogenesis of a number of diseases. ONE is an α,β ‐unsaturated aldehyde that can react in multiple ways and with glutathione, proteins and DNA. Heavy isotope‐labeled analogs of ONE are not readily available for conducting mechanistic studies or for use as internal standards in mass spectrometry (MS)‐based assays. An efficient one‐step cost‐effective method has been developed for the preparation of C‐9 deuterium‐labeled ONE. In addition, a method for specific deuterium labeling of ONE at C‐2, C‐3 or both C‐2 and C‐3 has been developed. This latter method involved the selective reduction of an intermediate alkyne either by lithium aluminum hydride or lithium aluminum deuteride and quenching with water or deuterium oxide. The availability of these heavy isotope analogs will be useful as internal standards for quantitative studies employing MS and for conducting mechanistic studies of complex interactions between ONE and DNA bases as well as between ONE and proximal amino acid residues in peptides and proteins. Copyright © 2011 John Wiley & Sons, Ltd.     

An antibody‐free strategy for screening putative HDM2 inhibitors using crude bacterial lysates expressing GST‐HDM2 recombinant protein

Targeting the interaction of p53 with its natural inhibitor MDM2 by the use of small synthetic molecules has emerged as a promising pharmacological approach to restore p53 oncosuppressor function in cancers retaining wild‐type p53. The first critical step in the experimental validation of newly synthesized small molecules developed to inhibit MDM2‐p53 interaction is represented by the evaluation of their efficacy in preventing the formation of the MDM2‐p53 complex. This can be achieved using the in vitro reconstructed recombinant MDM2‐p53 complex in cell‐free assays. A number of possible approaches have been proposed, which are however not suitable for screening large chemical libraries, due to the high costs of reagents and instrumentations, or the need of large amounts of highly pure recombinant proteins. Here we describe a rapid and cheap method for high‐throughput screening of putative inhibitors of MDM2‐p53 complex formation – based on the use of GST‐recombinant proteins – that does not require antibodies and recombinant protein purification steps from bacterial cell lysates. Copyright © 2013 John Wiley & Sons, Ltd.     

Role of Bcl‐2 in tumour cell survival and implications for pharmacotherapy

Objectives Bcl‐2 is a protein that inhibits apoptosis, leading to cell survival. The Bcl‐2 family has six different anti‐apoptotic proteins, three pro‐apoptotic proteins that are similar in structure, and other integrating proteins that function as promotors or inhibitors in the progression of apoptosis. In this discussion paper, we provide an overview of apoptosis, the role of Bcl‐2 in normal cellular and molecular processes, and the role of Bcl‐2 in tumour cell survival. It focuses primarily on anti‐apoptotic Bcl‐2, its activation in cancer, the manner in which it regulates the intrinsic and extrinsic mechanisms of apoptosis, and its broad molecular interactions with other critical proteins in the cell. Certain cancer treatments are reviewed and related directions for the future are presented. Key findings Apoptosis is common to all organisms – for eukaryotes it is a normal process of development and regeneration. The rate at which apoptosis occurs is critical to the survival of the organism, as too much can lead to the onset of degenerative diseases such as dementia, and too little may lead to cancer. FKBP‐38 is a binding protein that has been discovered to be upregulated in highly aggressive cancers and binds to Bcl‐2 rather than the pro‐apoptotics to induce a state of hyper‐mitosis. A short binding protein (Nur‐77) provides new insights into Bcl‐2 ‘masking’. Nurr‐77 binds to Bcl‐2 and exposes the BH3 domain, transforming it from a cancer promoter to an unorthodox cancer inhibitor. This presents in itself an interesting and exciting opportunity – increasing the rate of apoptosis in neoplastic cells that are usually protected by Bcl‐2 activity at the mitochondria. Summary Development of drugs in the form of BH3‐only and BH123 mimetic drugs provide a interesting avenue for cancer therapy for the future. Drugs that can either promote, or mimic anti‐IAP activity such as Smac/Diablo would certainly be productive, thereby inducing apoptosis. Medicinal usage which can effectively suppress FKBP38 in Bcl‐2‐dependent cancers would provide further arsenal to combat apoptotic irregularities, particularly a treatment that is more dominant than kinetin riboside. WAVE‐1 inhibitors may effectively suppress the phosphorylation of Bcl‐2, thereby potentially reducing hyper‐mitosis and increasing apoptosis. Recent findings shed molecular light on PDT, namely ER stress, and potential for anti‐cancer therapy via either apoptosis or autophagy. A drug that can effectively upregulate Nurr‐77, thereby masking the anti‐apoptotic properties of Bcl‐2, would indeed be life‐saving for cancer patients.     

The crude extract of Corni Fructus inhibits the migration and invasion of U‐2 OS human osteosarcoma cells through the inhibition of matrix metalloproteinase‐2/‐9 by MAPK signaling

Osteosarcoma is the most common primary malignancy of the bone cancers. In the Chinese population, the crude extract of Corni Fructus (CECF) has been used as Traditional Chinese medicine to treat several different diseases for hundreds of years. In the present study, effects of CECF on inhibition of migration and invasion in U‐2 OS human osteosarcoma cells were examined. CECF significantly inhibited migration and invasion of U‐2 OS human osteosarcoma cells. We also found that CECF inhibited activities of matrix metalloproteinases‐2 (MMP‐2) and matrix metalloproteinases‐9 (MMP‐9). CECF decreased protein levels of FAK, PKC, SOS1, MKK7, MEKK3, GRB2, NF‐κB p65, COX‐2, HIF‐1α, PI3K, Rho A, ROCK‐1, IRE‐1α, p‐JNK1/2, p‐ERK1/2, p‐p38, Ras, p‐PERK, MMP‐2, MMP‐9, and VEGF in U‐2 OS cells. Results of this study indicate that CECF may have potential as a novel anticancer agent for the treatment of osteosarcoma by inhibiting migration and invasion of cancer cells © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 53–63, 2015.     

Expression of pro‐inflammatory cytokines and mediators induced by Bisphenol A via ERK‐NFκB and JAK1/2‐STAT3 pathways in macrophages

Macrophages not only play an important role in the innate immune response but also participate in many inflammatory and infectious diseases including asthma, diabetes, obesity, cardiovascular diseases, and cancers. Bisphenol A (BPA) is the most commonly used component for plastic products. However, BPA is an endocrine disruptor for mammals and participates in several inflammatory and infectious diseases. Up until now, there are no researches demonstrated the potential role of BPA in macrophage activation and its relative mechanism. BPA promoted the generation of proinflammatory cytokines IL‐1β, IL‐6, and TNFα in a concentration‐dependent manner (P < 0.05). BPA was identified to increase the expression of proinflammatory mediators NO and PGE2, and its upstream factors iNOS, COX2, and cPLA2 in a concentration‐dependent manner (P < 0.05). Phosphorylation and nuclear translocation of NF‐κB p65 were significantly induced by BPA via IκB degradation (P < 0.05). In addition, phosphorylation of ERK significantly induced by BPA at a concentration which was less than that for phosphorylation of p38 MAPK and JNK (P < 0.05). Furthermore, phosphorylation of STAT3 significantly induced by BPA at a concentration lower than that for phosphorylation of STAT1 (P < 0.05). Phosphorylation of JAK1 and JAK2 was also significantly induced by BPA in a concentration‐dependent manner (P < 0.05).     

Exploring the RNA‐bound and RNA‐free human Argonaute‐2 by molecular dynamics simulation method

Argonaute 2 (Ago2) protein is the major vehicle of microRNAs (miRNAs)‐guided gene repression and silencing processes. Although the crystal structure of human Ago2 (hAgo2) has recently been disclosed, the information of dynamically structural character of protein–RNA recognition is still lacking. Molecular dynamics simulations were used to systematically explore hAgo2 in the presence and absence of RNA duplex. Stable direct and water‐mediated hydrogen bonds were observed between guide RNA backbone atoms and hAgo2, especially for nucleotides 2–7. In addition, water‐mediated hydrogen bonds are indicated to be critical in the specific recognition between hAgo2 and the conserved adenine in position 1 of target RNA. The core domains (N, PAZ, MID, and PIWI) possess rigid body movements during the simulations. The motions of N‐PAZ and PIWI‐MID are negatively correlated with or without RNA binding and PAZ domain is identified as the most mobile domain in both systems. The reorientation of PAZ domain not only influences the binding of helix‐7 and RNA duplex, the initial pairing process, but also the shape of N‐PAZ cleft, where the supplemental base pairing occurs. It is speculated that PAZ domain could be a key regulator in hAgo2‐mediated miRNA‐induced gene regulation.     

Down‐regulation of KIF2A inhibits gastric cancer cell invasion via suppressing MT1‐MMP

Gastric cancer accounts for a sizeable proportion of global cancer mortality with high morbidity and poor prognosis. Kinesin superfamily proteins (KIFs) are microtubule‐dependent motor proteins that function as oncogenes in cancer cells, it has been discovered in recent years. Kinesin family member 2a (KIF2A), a member of the KIFs, has received attention for its role in carcinogenesis and its prognostic value in several human cancers such as breast cancer, colorectal cancer, and squamous cell carcinoma. However, the role of KIF2A in human gastric cancer remains unknown. In this study we aimed to explore the expression and biological functions of KIF2A in human gastric cancer cells, as well as to reveal its potential action mechanism. First, we found that KIF2A was markedly increased in gastric cancer cells (MKN‐28, MKN‐45, NCI‐N87 and SGC‐7901) compared to normal gastric mucosa epithelial cells (GES‐1). Then KIF2A was successfully silenced in MKN‐45 and SGC‐7901 cells to facilitate further research into its function. We discovered that KIF2A silencing can significantly inhibit the growth and invasion of MKN‐45 and SGC‐7901 cells in a time‐independent manner, accompanying a decreased expression of Membrane type 1‐matrix metalloproteinase (MT1‐MMP). When MT1‐MMP was reintroduced into MKN‐45 and SGC‐7901 cells in the KIF2A‐siRNA group, only invasion inhibition effects on MKN‐45 and SGC‐7901 cells induced by KIF2A silencing can be reversed. In conclusion, our study reveals that down‐regulation of KIF2A can inhibit gastric cancer cell invasion by suppressing MT1‐MMP.     

Convenient synthesis of 18F‐radiolabeled R‐(−)‐N‐n‐propyl‐2‐(3‐fluoropropanoxy‐11‐hydroxynoraporphine

Aporphines are attractive candidates for imaging D₂ receptor function because, as agonists rather than antagonists, they are selective for the receptor in the high affinity state. In contrast, D₂ antagonists do not distinguish between the high and low affinity states, and in vitro data suggests that this distinction may be important in studying diseases characterized by D₂ dysregulation, such as schizophrenia and Parkinson's disease. Accordingly, MCL‐536 (R‐(?)‐N‐n‐propyl‐2‐(3‐[¹⁸F]fluoropropanoxy‐11‐hydroxynoraporphine) was selected for labeling with ¹⁸F based on in vitro data obtained for the non‐radioactive (¹⁹F) compound. Fluorine‐18‐labeled MCL‐536 was synthesized in 70% radiochemical yield, >99% radiochemical purity, and specific activity of 167 GBq/µmol (4.5 Ci/µmol) using p‐toluenesulfonyl (tosyl) both as a novel protecting group for the phenol and a leaving group for the radiofluorination.     

(Z)‐2‐(3‐Chlorobenzylidene)‐3,4‐dihydro‐N‐(2‐methoxyethyl)‐3‐oxo‐2H‐benzo[b][1,4]oxazine‐6‐carboxamide as GSK‐3β inhibitor: Identification by virtual screening and its validation in enzyme‐ and cell‐based assay

Glycogen synthase kinase 3β (GSK‐3β) is a widely investigated molecular target for numerous diseases including Alzheimer's disease, cancer, and diabetes mellitus. The present study was aimed to discover new scaffolds for GSK‐3β inhibition, through protein structure‐guided virtual screening approach. With the availability of large number of GSK‐3β crystal structures with varying degree of RMSD in protein backbone and RMSF in side chain geometry, herein appropriate crystal structures were selected based on the characteristic ROC curve and percentage enrichment of actives. The validated docking protocol was employed to screen a library of 50,000 small molecules using molecular docking and binding affinity calculations. Based on the GLIDE docking score, Prime MMGB/SA binding affinity, and interaction pattern analysis, the top 50 ligands were selected for GSK‐3β inhibition. (Z)‐2‐(3‐chlorobenzylidene)‐3,4‐dihydro‐N‐(2‐methoxyethyl)‐3‐oxo‐2H‐benzo[b][1,4]oxazine‐6‐carboxamide (F389‐0663, 7 ) was identified as a potent inhibitor of GSK‐3β with an IC₅₀ value of 1.6 μm . Further, GSK‐3β inhibition activity was then investigated in cell‐based assay. The treatment of neuroblastoma N2a cells with 12.5 μm of F389‐0663 resulted in the significant increase in GSK‐3β Ser9 levels, which is indicative of the GSK‐3β inhibitory activity of a compound. The molecular dynamic simulations were carried out to understand the interactions of F389‐0663 with GSK‐3β protein.     

Structure‐based approaches for the design of benzimidazole‐2‐carbamate derivatives as tubulin polymerization inhibitors

Microtubules are highly dynamic assemblies of α/β‐tubulin heterodimers whose polymerization inhibition is among one of the most successful approaches for anticancer drug development. Overexpression of the class I (βI) and class III (βIII) β‐tubulin isotypes in breast and lung cancers and the highly expressed class VI (βVI) β‐tubulin isotype in normal blood cells have increased the interest for designing specific tubulin‐binding anticancer therapies. To this end, we employed our previously proposed model of the β‐tubulin–nocodazole complex, supported by the recently determined X‐ray structure, to identify the fundamental structural differences between β‐tubulin isotypes. Moreover, we employed docking and molecular dynamics (MD) simulations to determine the binding mode of a series of benzimidazole‐2‐carbamete (BzC) derivatives in the βI‐, βIII‐, and βVI‐tubulin isotypes. Our results demonstrate that Ala198 in the βVI isotype reduces the affinity of BzCs, explaining the low bone marrow toxicity for nocodazole. Additionally, no significant differences in the binding modes between βI‐ and βIII‐BzC complexes were observed; however, Ser239 in the βIII isotype might be associated with the low affinity of BzCs to this isotype. Finally, our study provides insight into the β‐tubulin–BzC interaction features essential for the development of more selective and less toxic anticancer therapeutics.     

环氧合酶-2及其抑制剂在胰腺癌血管靶向治疗中的作用

胰腺癌的诊治目前仍是医学界的难题,血管生成是胰腺癌生长和转移的必要因素。环氧合酶(cyclooxygenase,COX)为花生四烯酸代谢过程中的关键酶,存在COX-1及COX-2两种同工酶,其中COX-2在胰腺癌的发生发展及胰腺癌血管生成中发挥重要作用。COX-2抑制剂可分为非选择性及选择性两种,两种COX-2抑制剂都对胰腺癌及其血管形成的发生与进展存在抑制作用,COX-2抑制剂抗肿瘤血管形成的机制可能是抑制血管内皮生长因子(VEGF)、缺氧诱导因子-1及基质金属蛋白酶(MMP)的表达、降低前列腺素(PGs)及其他血管生成因子的表达、促进内皮细胞凋亡、抑制内皮细胞侵袭力和影响一氧化氮合酶(NOS)的表达。     

Synthesis,radiosynthesis and preliminary in vivo evaluation of [123I]‐(4‐fluorophenyl) {1‐[2‐(2‐iodophenyl)ethyl]piperidin‐4‐yl}methanone,a potential 5‐HT2A‐antagonist for SPECT brain imaging

Many people suffer from psychiatric illnesses like depression and anorexia. Relevant to these diseases is amongst others a malfunctioning of brain 5‐HT_2A‐receptors. To allow in vivo quantification of these receptors with Single Photon Emission Computerized Tomography (SPECT), a radiolabelled ligand with high 5‐HT_2A affinity is needed. This work reports the radiosynthesis of [¹²³I]‐(4‐fluorophenyl) {1‐[2‐(2‐iodophenyl)ethyl]piperidin‐4‐yl}methanone, the synthesis of its precursor, (4‐fluorophenyl) {1‐[2‐(2‐bromophenyl)ethyl]piperidin‐4‐yl}methanone, and the preliminary in vivo evaluation of the tracer. The precursor was synthesized with a total yield of 40%. Radiolabelling was performed using a halogen exchange reaction and the yield was 70%. Radiochemical purity was >95%, and specific activity was at least 2.4 Ci/µmol. Log P was measured to be 2.52. The tracer showed uptake in mice brain (3.5% I.D./g tissue at 3 min post injection) and therefore will be evaluated further by regional brain biodistribution and displacement studies in rabbits. Copyright © 2004 John Wiley & Sons, Ltd.     

Lupeol alters ER stress‐signaling pathway by downregulating ABCG2 expression to induce Oxaliplatin‐resistant LoVo colorectal cancer cell apoptosis

Colorectal cancer (CRC) is one of the most common cancers and causes of cancer‐related death. There are several first‐line chemotherapeutic drugs used to treat CRC. Oxaliplatin (OXA) is an alkylating cytotoxic agent that is usually combined with other chemotherapeutic drugs to treat stage II and stage III CRC. However, cancer cells commonly acquire multidrug resistance (MDR), which is a major obstruction to cancer treatment. Recent studies have shown that natural components from traditional Chinese medicine or foods that have many biological functions may be new adjuvant therapies in clinical trials. We challenged LoVo CRC cell lines with OXA in a dose‐dependent manner to create an OXA‐resistant model. The expression of ABCG2 was significantly higher, and levels of endoplasmic reticulum (ER) stress markers were lower than those Parental cells. However, Lupeol, which is found in fruits and vegetables, has been shown to have bioactive properties, including anti‐tumor properties that are relevant to many diseases. In our study, Lupeol downregulated cell viability and activated cell apoptosis. Moreover, Lupeol decreased the expression of ABCG2 and activated ER stress to induce OXA‐resistant cell death. Importantly, the anti‐tumor effect of Lupeol in OXA‐resistant cells was higher than that of LoVo Parental cells. In addition, we also confirmed our results with a xenograft animal model, and the tumor size significantly decreased after Lupeol injections. Our findings show that Lupeol served as a strong chemoresistant sensitizer and could be a new adjuvant therapy method for chemoresistant patients.     

Detection and identification of 2‐nitro‐morphine and 2‐nitro‐morphine‐6‐glucuronide in nitrite adulterated urine specimens containing morphine and its glucuronides

In vitro urine adulteration is a well‐documented practice adopted by individuals aiming to evade detection of drug use, when required to undergo mandatory sports and workplace drug testing. Potassium nitrite is an effective urine adulterant due to its oxidizing potential, and has been shown to mask the presence of many drugs of abuse. However, limited research has been conducted to understand its mechanism of action, and to explore the possibility of the drugs undergoing direct oxidation to form stable reaction products. In this study, opiates including morphine, codeine, morphine‐3‐glucuronide and morphine‐6‐glucuronide were exposed to potassium nitrite in water and urine to mimic the process of nitrite adulteration. It was found that two stable reaction products were detected by liquid chromatography‐mass spectrometry (LC‐MS) when morphine and morphine‐6‐glucuronide were exposed to nitrite. Isolation and elucidation using spectrometric and spectroscopic techniques revealed that they were 2‐nitro‐morphine and 2‐nitro‐morphine‐6‐glucuronide, respectively. These reaction products were also formed when an authentic morphine‐positive urine specimen was fortified with nitrite. 2‐Nitro‐morphine was found to be stable enough to undergo the enzymatic hydrolysis procedure and also detectable by gas chromatography‐mass spectrometry (GC‐MS) after forming a trimethylsilyl derivative. On the contrary, morphine‐3‐glucuronide did not appear to be chemically manipulated when exposed to potassium nitrite in urine. These reaction products are not endogenously produced, are relatively stable and can be monitored with both LC‐MS and GC‐MS confirmatory techniques. As a result, these findings have revealed the possibility for the use of 2‐nitro‐morphine and 2‐nitro‐morphine‐6‐glucuronide as markers for the indirect monitoring of morphine and morphine‐6‐glucuronide in urine specimens adulterated with nitrite. Copyright © 2013 John Wiley & Sons, Ltd.     

Genotoxicity evaluation of two kinds of smoke‐water and 3,7‐dimethyl‐2H‐furo[2,3‐c]pyran‐2‐one

Smoke, smoke‐water and aerosols have a stimulatory effect on seed germination and growth vigour of many seedlings, making them potentially useful for different purposes, provided they do not pose a health risk. Therefore, the genotoxicity of two kinds of smoke‐water and 3,7‐dimethyl‐2H‐furo[2,3‐c]pyran‐2‐one, a variant of the most active smoke compound (3‐methyl‐2H‐furo[2,3‐c]pyran‐2‐one) was evaluated using the Vitotox? assay. Smoke‐water extracts were obtained from burning leaves: Themeda triandra (smoke‐water Tt) and a mix of Themeda triandra and Passerina vulgaris (smoke‐water Kb). No genotoxic effect was observed for any of the three samples. However, the three samples are toxic at the highest concentrations (3,7‐dimethyl‐2H‐furo[2,3‐c]pyran‐2‐one, 2 ppm; smoke‐water Tt, dilutions 1 : 1, 1 : 2, 1 : 4; smoke‐water Kb, dilution 1 : 1) without addition of S9 mix. Both the butenolide 3,7‐dimethyl‐2H‐furo[2,3‐c]pyran‐2‐one and smoke‐water Tt are also toxic at high doses in the presence of S9 (2 ppm and dilutions 1 : 1 and 1 : 2, respectively), but not smoke‐water Kb. Thus, from these results, no genotoxicity of these three samples can be assumed, which is accordance with the previous tests performed with 3‐methyl‐2H‐furo[2,3‐c]pyran‐2‐one and a smoke‐water. Copyright © 2010 John Wiley & Sons, Ltd.