Ozone exposure in the culture medium inhibits enterovirus 71 virus replication and modulates cytokine production in rhabdomyosarcoma cells

In the present study, the effects of ozone exposure on enterovirus 71 (EV71) replication and related cytokine production were investigated. Rhabdomyosarcoma cells (RD) were exposed to 0.5, 1, 1.5 and 2 ppm ozone or filtered air under different exposure regimens before or after infection for 1 or 2 h. The results revealed that at a proper concentration of ozone, e.g., 1.5 or 2 ppm, ozone exposure restricted virus production, prolonged survival time of cells and modulated cytokine production related to EV71 infection. Upon exposure of non-infected cells to ozone at 1.5 or 2 ppm for 1h, the production of IL-1beta, IL-6 and TNF-alpha was primed and boosted by the subsequent EV71 infection, generating an inhibitory effect on EV71 replication during the post-infection period of 48 h. While infected cells were exposed to ozone for 2 h at 1.5 or 2 ppm, ozone did not affect cytokine production by RD cells in response to EV71 infection. The data showed that ozone effect on induction of cytokine was only found in uninfected cells. The ozone-induced cytokines produced prior to the onset of EV71 infection generated antiviral effects, which proved beneficial in suppressing the subsequent EV71 infection.     

Ozone exposure in the culture medium inhibits enterovirus 71 virus replication and modulates cytokine production in rhabdomyosarcoma cells

In the present study, the effects of ozone exposure on enterovirus 71 (EV71) replication and related cytokine production were investigated. Rhabdomyosarcoma cells (RD) were exposed to 0.5, 1, 1.5 and 2 ppm ozone or filtered air under different exposure regimens before or after infection for 1 or 2 h. The results revealed that at a proper concentration of ozone, e.g., 1.5 or 2 ppm, ozone exposure restricted virus production, prolonged survival time of cells and modulated cytokine production related to EV71 infection. Upon exposure of non-infected cells to ozone at 1.5 or 2 ppm for 1 h, the production of IL-1β, IL-6 and TNF-α was primed and boosted by the subsequent EV71 infection, generating an inhibitory effect on EV71 replication during the post-infection period of 48 h. While infected cells were exposed to ozone for 2 h at 1.5 or 2 ppm, ozone did not affect cytokine production by RD cells in response to EV71 infection. The data showed that ozone effect on induction of cytokine was only found in uninfected cells. The ozone-induced cytokines produced prior to the onset of EV71 infection generated antiviral effects, which proved beneficial in suppressing the subsequent EV71 infection.     

Fullerene-based amino acid nanoparticle interactions with human epidermal keratinocytes

The functionalization of C₆₀ with such complexes as amino acids has the potential to provide greater interaction between the fullerene and the biological environment yielding potential new medical and pharmacological applications. Although scientific research in the past decade has revealed much about the chemical and physical properties of C₆₀, the biological activities of this compound and its derivatives are still relatively unclear. In an attempt to understand the biological activity of functionalized C₆₀, human epidermal keratinocytes (HEK) were exposed to fullerene-based amino acid (Baa) solutions ranging in concentrations of 0.4–0.00004 mg/mL in a humidified 5% CO₂ atmosphere at 37 °C. MTT cell viability after 48 h significantly decreased (p < 0.05) for concentrations of 0.4 and 0.04 mg/mL. In an additional study, human cytokines IL-6, IL-8, TNF-, IL-1β, and IL-10 were assessed for concentrations ranging from 0.4–0.004 mg/mL. Media was harvested at 1, 4, 8, 12, 24 and 48 h for cytokine analysis. IL-8 concentrations for the 0.04 mg/mL treatment were significantly greater (p < 0.05) than all other concentrations at 8, 12, 24, and 48 h. IL-6 and IL-1β activities were greater at the 24 h and 48 h for 0.4 and 0.04 mg/mL. No significant TNF- or IL-10 activity existed at any time points for any of the concentrations. These results indicate that concentrations lower than 0.04 mg/mL initiate less cytokine activity and maintain cell viability. In HEK, Baa concentrations of 0.4 and 0.04 mg/mL decrease cell viability and initiate a pro-inflammatory response.     

微量细胞培养法在手足口病毒研究中的应用

目的建立应用于手足口病毒分离和对手足口EV71病毒抑制药物筛选的微量细胞培养方法。方法比较微量细胞培养结合real-timePCR和直接应用real-timePCR两种方法检测手足口病毒的差别,并应用微量细胞培养采用细胞病变效应法和MTT分析法,观察利巴韦林对EV71病毒的抑制作用。结果经微量细胞培养后EV71、CA16阳性检出率为90%和80%,但PE阴性样本20%检测为EV71阳性;利巴韦林浓度为0.4、0.2、0.1mg/mL时对EV71病毒有抑制作用,抑制率分别为30.28%、28.09%和29.16%。结论微量细胞培养在手足口病毒分离检测和对其抑制作用药物筛选研究中具有可行性,且其具有操作简便、省时省力等优点。     

Effects of in vitro ozone exposure on peroxidative damage, membrane leakage, and taurine content of rat alveolar macrophages

Rat alveolar macrophages (AM) were isolated by pulmonary lavage, allowed to adhere to a tissue culture flask, and then exposed to 0.45 +/- 0.05 ppm ozone. After exposures ranging from 0 to 60 min, the medium was decanted and cells were harvested. Cells were assayed for oxidant damage and media analyzed for leakage of intracellular components. Increasing length of exposure to ozone resulted in a decreased number of adherent AM and decreased cell viability. Resting and zymosan-stimulated chemiluminescence increased immediately after ozone exposure and reached a maximum at 15-30 min, then declined to initial levels after 60 min of ozone exposure. Lipid peroxidation and leakage of protein and K+ ions increased with increasing length of exposure to ozone, while leakage of reduced and oxidized glutathione increased through 30 min, then declined (reduced) or leveled off (oxidized). Activity of the Na+/K+ ATPase decreased with time while intracellular taurine concentration exhibited an initial rise, peaked at 30 min, and then returned to the untreated level. Leakage of taurine into the medium increased with time of exposure, suggesting that exposure of AM to ozone results in a shift from bound to free intracellular taurine. These data indicate that in vitro exposure of AM to ozone results in a time-dependent alteration of cell function, membrane integrity, and viability.     

Effect of microcystin-LR on protein phosphatase activity and glycogen content in isolated hepatocytes of fed and fasted juvenile goldfish Carassius auratus L.

Toxic effects of microcystin-LR were studied in hepatocytes isolated from fed and fasted juvenile goldfish Carassius auratus (30 g body weight). The hepatocytes were incubated with 10 μg MC-LR l⁻¹ during 4 h. MC-LR induces no effect in terms of cell number and viability. The toxin accumulation pattern was different in fed and fasted treatments. MC-LR accumulated more rapidly in ‘fasted’ cells where the highest concentration was observed by 1 h of exposure. It was delayed to 4 h in the ‘fed’ cells.

MC-LR accumulation induced a severe decrease in hepatic protein phosphatase activity in both treatments. It was almost totally inhibited in both treatments during the first hour of exposure. The glycogen content was significantly reduced after 2 h of exposure in the fasting treatments, but not in the feeding one.     

我国肠道病毒71型（EV71）疫苗株生物信息学分析

目的 比较3个EV71疫苗株病毒的生物信息学特点,为疫苗的质控和探讨该病毒疫苗的免疫机制提供参考.方法 本研究利用DNAstar MegAlign、DNAMAN Alignment、ANtheProt等生物学软件,比较了我国已进入临床试验的3家EV71全病毒灭活疫苗毒株的基因组及氨基酸序列,并对英衣壳蛋白二级结构及可能存在的抗原表位进行了预测分析.结果 3个疫苗株病毒基因同源性为97％～99％;氨基酸同源性为98％～99％;二级结构一致率在95％以上;均存在35个潜在抗原表位区域.结论 3个疫苗株生物信息学分析结果存在高度的一致性,提示疫苗具有相似的抗原性和免疫原性.     

5种不同细胞对柯萨奇病毒A组16型和肠道病毒71型的敏感性研究

 目的   评价肠道病毒研究中常用的5种细胞对柯萨奇病毒A组16型（coxsackievirus A16,CA16）和肠道病毒71型（enterovirus 71,EV71）的敏感性,为分离和培养CA16和EV71提供实验室依据。方法  用分离的各15株CA16和EV71分别感染RD、Vero、KMB17、Hep2和L20B细胞,每天观察细胞病变情况,按Karber法计算病毒感染性滴度。采用不同的统计学方法（包括方差分析和卡方检验）比较5种细胞对CA16和EV71的敏感性差异。 结果   5种细胞的CA16和EV71病毒感染性滴度间的差异具有统计学意义（CA16：F＝18.481,P＝0.000;EV71：χ²＝63.106,P＝0.000）。在5种细胞中,RD细胞对2种病毒的敏感性最高,CA16和EV71的病毒感染性滴度分别可达7.8和8.2 lgCCID₅₀/ml,且均于接种细胞后48 h达增殖高峰,第4天进入增殖平台期;2种病毒在Hep2和L20B细胞中的病毒感染性滴度均较低,CA16分别为5.2和4.2 lgCCID₅₀/ml,EV71分别为4.9和4.0 lgCCID₅₀/ml,且2种病毒在接种后第6~7天才达增殖高峰,但仅L20B细胞中的2种病毒于接种后第7天进入增殖平台期;CA16在Vero和KMB17细胞中的病毒感染性滴度分别为6.9和7.3 lgCCID₅₀/ml,EV71分别为7.1和6.8 lgCCID50/ml,2种病毒均在接种Vero和KMB17细胞后第3天达到增殖高峰,第5天进入增殖平台期。 结论   RD细胞对CA16和EV71的敏感性最好,其次是Vero和KMB17细胞,而Hep2和L20B细胞并非是分离和培养这2种病毒的理想细胞。     

Inhibition of enterovirus 71 replication by chrysosplenetin and penduletin

In recent years, enterovirus 71 (EV71) infections have caused an increasing epidemic in young children, accompanying with more severe nervous system disease and more deaths. Unfortunately, there is no specific medication for it so far. Here we investigated the anti-EV71 activity of chrysosplenetin and penduletin, two o-methylated flavonols isolated from the leaves of Laggera pterodonta. These two compounds were found to have strong activity in vitro against EV71 with low cytotoxicity. In the cytopathic effect (CPE) inhibition assays, both plaque reduction assay and virus yield inhibition assay, the compounds showed a similar 50% inhibitory concentration (IC(50)) value of about 0.20 μM. The selectivity indices (SI) of chrysosplenetin and penduletin were 107.5 and 655.6 in African green monkey kidney (Vero) cells, and 69.5 and 200.5 in human rhabdomyosarcoma (RD) cells, accordingly. The preliminary mechanism analysis indicates that they function not through blocking virus entry or inactivating virus directly but inhibiting viral RNA replication. In the time-of-addition assay, both compounds inhibited progeny virus production and RNA replication by nearly 100% when introduced within 4h post infection. In addition to EV71, both compounds inhibited several other human enteroviruses with similar efficacy. These findings provide a significant lead for the discovery of anti-EV71 drug.     

人用H5N1禽流感病毒疫苗生产工艺的优化

目的 建立优化的人用H5N1禽流感病毒疫苗生产的工艺。方法 在不同的稀释倍数、收获时间及灭活剂添加量下,通过测量收获液的病毒滴度和血凝效价,来确定病毒的最佳生产条件,并对离心法和凝胶过滤层析法的纯化效果进行对比。结果 103~104半数鸡胚感染量(50% egg infective dose,EID50)病毒接种鸡胚,收获的鸡胚尿囊液的病毒滴度和血凝效价最高,分别为10-8.3EID50和1∶480;在56~72 h血凝效价最高。甲醛浓度1∶10 000灭活144 h为灭活最佳条件。两种纯化方法得到的样品纯度和卵清蛋白的去除率相近,但离心纯化法和凝胶过滤层析纯化法病毒回收率有较大的差异,分别为19%和70%。结论 成功建立了高产毒的鸡胚基质H5N1禽流感病毒培养、灭活及纯化工艺。     

Gene expression and estrogen sensitivity in rat uterus after developmental exposure to the polybrominated diphenylether PBDE 99 and PCB

Considering the presence of polybrominated diphenylethers (PBDEs) in human milk and cord blood, and the estrogenic activity of some congeners, it is conceivable that PBDEs may interact with developing neuroendocrine systems. We investigated effects of 2,2′,4,4′,5-pentabromo-DE (PBDE 99), a major congener in human milk, on development of brain and reproductive organs, with focus on estrogen target gene expression. Time-pregnant Long Evans rats were subcutaneously injected with PBDE 99 (1 or 10 mg/kg/day), the PCB mixture Aroclor 1254 (10 mg/kg/day), known to interfere with sexual development, or vehicle, from gestational day (GD) 10 to GD 18. In female offspring, anogenital distance was unaffected by PBDE 99 but increased by Aroclor; puberty (vaginal opening) was not significantly changed. Adult PBDE 99-exposed offspring exhibited unchanged uterine weight but increased ovarian weight. Uterine mRNA levels of estrogen target genes were determined by real-time PCR. Progesterone receptor (PR) mRNA was down-regulated at both PBDE 99 doses, estrogen receptor alpha (ER alpha), ER beta and insulin-like growth factor-I (IGF-I) were up-regulated at the lower dose. Aroclor induced different effect patterns. In order to investigate possible changes in sensitivity of target genes to estrogen, some offspring were ovariectomized at 10 weeks of age, s.c. injected with estradiol-17β (E2, 10 μg/kg) or vehicle at 12 weeks, and sacrificed 6 h later. PBDE 99 dose-dependently reduced the magnitude of IGF-I mRNA induction by E2, and increased the magnitude of ER beta repression. PBDE 99 also influenced baseline levels of PR, IGF-I and ER beta mRNAs in ovariectomized, vehicle-injected controls. These data indicate that developmental exposure to PBDE 99 at doses devoid of general toxicity, affects the regulation of estrogen target genes in uterus. Since PBDE 99 was detected in blood and adipose tissue of adult offspring, these effects may result from interactions with developmental processes, adult functions, or a combination of both.     

Chelerythrine and dihydrochelerythrine induce G1 phase arrest and bimodal cell death in human leukemia HL-60 cells

A quaternary benzo[c]phenanthridine alkaloid chelerythrine displays a wide range of biological activities including cytotoxicity to normal and cancer cells. In contrast, less is known about the biological activity of dihydrochelerythrine, a product of chelerythrine reduction. We examined the cytotoxicity of chelerythrine and dihydrochelerythrine in human promyelocytic leukemia HL-60 cells. After 4 h of treatment, chelerythrine induced a dose-dependent decrease in the cell viability with IC₅₀ of 2.6 μM as shown by MTT reduction assay. Dihydrochelerythrine appeared to be less cytotoxic since the viability of cells exposed to 20 μM dihydrochelerythrine for 24 h was reduced only to 53%. Decrease in the viability induced by both alkaloids was accompanied by apoptotic events including the dissipation of mitochondrial membrane potential, activation of caspase-9 and -3, and appearance of cells with sub-G1 DNA. Moreover, chelerythrine, but not dihydrochelerythrine, elevated the activity of caspase-8. A dose-dependent induction of apoptosis and necrosis by chelerythrine and dihydrochelerythrine was confirmed by annexin V/propidium iodide dual staining flow cytometry. Besides, both alkaloids were found to induce accumulation of HL-60 cells in G1 phase of the cell cycle. We conclude that both chelerythrine and dihydrochelerythrine affect cell cycle distribution, activate mitochondrial apoptotic pathway, and induce apoptosis and necrosis in HL-60 cells.     

Activity of telithromycin against erythromycin-susceptible and -resistant Streptococcus pneumoniae isolates from adults with invasive infections

A telithromycin (TEL) kill-kinetics study was conducted with 120 clinically significant Streptococcus pneumoniae isolates (60 susceptible and 60 highly resistant to erythromycin). Time–kill curves were performed using different antibiotic concentrations. The minimum inhibitory concentrations (MICs) of TEL were low for both erythromycin-susceptible (MIC ≤ 0.016 mg/L) and erythromycin-resistant strains (MIC ≤ 0.25 mg/L). TEL showed 99.9% killing of all erythromycin resistant strains at 18–24 h of incubation. Even for strains with erythromycin MICs ≥ 64.0 mg/L, TEL was uniformly bactericidal at 0.25 mg/L.     

Effects of binary mixtures of xenoestrogens on gonadal development and reproduction in zebrafish

Previous studies exposing fish to xenoestrogens have demonstrated vitellogenin (VTG) induction, delayed gametogenesis, altered sex ratio, and decreased reproductive performance, with a majority of those studies focusing on exposure to single chemicals. The objective of this study was to determine the effects of binary mixtures of a weak estrogen receptor agonist, nonylphenol (NP) and a potent estrogen receptor agonist, 17-ethinylestradiol (EE) on sex ratios, gametogenesis, VTG induction, heat shock protein 70 (HSP70) expression and reproductive capacity in zebrafish (Danio rerio). Fish were exposed from 2 to 60 days post-hatch (dph) to nominal concentrations of 10 or 100 μg/l NP (NP10 or NP100, respectively), 1 or 10 ng/l EE (EE1 or EE10, respectively), 1 ng/l EE + 10 or 100 μg/l NP (EE1 + NP10 or EE1 + NP100, respectively), 10 ng/l EE + 10 or 100 μg/l NP (EE10 + NP10 or EE10 + NP100, respectively) or solvent control (0.01% acetone, v/v) in a static-renewal system with replacement every 48 h. At 60 dph, fish from each treatment were euthanized for histological examination of gonads, and whole body VTG and HSP70 levels. Remaining fish were reared in clean water until adulthood (240 dph) for breeding studies. In all EE10 exposure groups (EE10, EE10 + NP10 and EE10 + NP100), increasing NP concentration acted antagonistically to the action of EE in terms of VTG induction at 60 dph. Similarly, non-additivity was observed with egg production, where EE1 + NP100 exposure resulted in significantly more eggs produced per breeding trial than EE1 alone. Histological staging of oogenesis revealed suppressed gametogenesis in an additive fashion in females at 60 dph. There were no differences among treatment groups in whole body HSP70 expression in 60 dph fish or in gonadal HSP70 expression in adult fish. Although there was no statistical evidence of non-additivity, breeding trials in adults revealed significant reductions in egg viability, egg hatchability and/or F₁ swim-up success, suggesting that developmental exposures to xenoestrogens may cause irreversible effects on egg quality and progeny even after periods of depuration. In conclusion, these results suggest that environmentally relevant mixtures of NP and EE can produce additive or non-additive effects that depend on the particular response being determined and the respective exposure concentrations of each chemical.     

A hand-held apparatus for “nose-only” exposure of mice to inhalable microparticles as a dry powder inhalation targeting lung and airway macrophages

Microparticles containing isoniazid and rifabutin were aerosolised using a simple apparatus fabricated from a 15-ml centrifuge tube. The dose available for inhalation by rodents was determined by collecting microparticles emitted at the delivery port. The dose available for inhalation was proportional to durations of exposure ranging from 10 to 90 s (10.5–13.5 CV%) and the weight of powder taken for fluidization (10–50 mg, r² = 0.982). The apparatus was then used to administer inhalations of microparticles to mice. Other groups of mice received free rifabutin orally, or by i.v. injection. Rifabutin was estimated in serum and tissues of dosed mice by HPLC. When 20 mg of microparticles were loaded in the apparatus, 2.5 mg were collected at the delivery port in 30 s of operation. Mice inhaled 300 μg of the 2.5 mg emitted at the delivery port. Airway and lung macrophages of mice receiving inhalations for 30 s accumulated 0.38 μg of rifabutin, while the amount in blood serum of these mice was 0.62 μg. In mice receiving 83 μg rifabutin i.v. or orally, the intracellular amounts were 0.06 and 0.07 μg respectively, while the amounts in serum were 1.02 and 0.80 μg. These observations confirmed that inhalation of microparticles targeted airway and lung macrophages.     

Synthesis and transdermal properties of acetylsalicylic acid and selected esters

The primary aim of this study was to determine the transdermal penetration of acetylsalicylic acid and some of its derivatives, to establish a correlation, if any, with selected physicochemical properties and to determine if transdermal application of acetylsalicylic acid and its derivatives will give therapeutic drug concentrations with respect to transdermal flux. Ten derivatives of acetylsalicylic acid were prepared by esterification of acetylsalicyloyl chloride with ten different alcohols. The experimental aqueous solubility, log D and transdermal flux values were determined for acetylsalicylic acid and its derivatives at pH 4.5. In vitro penetration was measured through excised female human abdominal skin in diffusion cells. The experimental aqueous solubility of acetylsalicylic acid (6.56 mg/ml) was higher than that of the synthesised acetylsalicylate derivatives (ranging from 1.76 × 10⁻³ to 3.32 mg/ml), and the log D of acetylsalicylic acid (−0.85) was lower than that of its derivatives (ranging from −0.25 to 1.95). There was thus an inverse correlation between the aqueous solubility data and the log D values. The experimental transdermal flux of acetylsalicylic acid (263.83 nmol/cm² h) was much higher than that of its derivatives (ranging from 0.12 to 136.02 nmol/cm² h).     

An approach to As(III) and As(V) bioavailability studies with Caco-2 cells

Foods and drinking water are the main sources of human exposure to inorganic arsenic [As(III) and As(V)]. After oral ingestion, the intestinal epithelium is the first barrier to absorption of these species. A human intestinal cell line (Caco-2) was used to evaluate cell retention and transport of As(III) (15.6–156.0 μM) and/or As(V) (15.4–170.6 μM). Cell monolayer integrity, cell viability, membrane damage and effects on cell metabolism were evaluated. Only the highest concentrations assayed [As(III): 156.0 μM; As(V): 170.6 μM] produced a cytotoxic effect with different cellular targets: As(III) altered the permeability of tight junctions, and As(V) caused uncoupling of the respiratory chain. Retention and transport of As(III) was more efficient than that of As(V). After 4 h of exposure to As(III) or As(V), monolayer retention percentages varied between 0.87–2.28% and 0.14–0.39%, respectively. Transepithelial transport was greater for As(III) (5.82–7.71%) than for As(V) (not detectable—1.55%). The addition of As(III) and As(V) jointly produced a transport rate similar to that observed when they were added independently.     

Effect of ozone and histamine on airway permeability to horseradish peroxidase in guinea pigs

Airway permeability was studied in groups of male guinea pigs at 2, 8, and 24 h after a 1-h exposure to 1 ppm ozone or at 2 h after a 1-h exposure to filtered air (control). Intratracheal administration of 2 mg horseradish peroxidase (HRP) was followed by blood sampling at 5-min intervals up to 30 min. The rate of appearance of HRP in plasma was significantly higher at 2 and 8 h after ozone exposure than that found in animals examined 2 h after air exposure or 24 h after ozone exposure. A dose of 0.12 mg/kg of subcutaneous histamine given after the 15 min blood sample significantly increased the already elevated permeability seen at 2 h post ozone, but had no effect on animals exposed to filtered air 2 h earlier or to ozone 24 h earlier. No difference was seen in the amount of subcutaneous radiolabeled histamine in the lungs of animals exposed 2 h earlier either to air or to ozone. These data indicate that a short-term exposure to ozone produced a reversible increase in respiratory epithelial permeability to HRP in guinea pigs. The potentiation of this increased permeability by histamine may be another manifestation of ozone-induced hyperreactivity.     

Developmental and reproductive toxicity evaluation of toluene vapor in the rat II. Developmental toxicity

The developmental toxicity of toluene was evaluated via whole body inhalation exposure, in pregnant Sprague Dawley rats exposed to toluene (99.9% pure) from gestation day (GD) 6–15 inclusive, 6 h/day, at concentrations of 0, 250, 750, 1500 and 3000 ppm (0, 938, 2812, 5625 and 11250 mg/m³). Doses were selected from a preliminary study performed over a range of concentrations from 0 to 5000 ppm, in which maternal and fetal toxicity were observed at 2000 ppm and above. This study has been cited in various regulatory documents and is presented here to allow greater accessibility to results and conclusions.

Toluene induced clinical signs in pregnant dams (ataxia, hyper-responsivity, increased water intake, decreased food consumption) at 3000 ppm, ataxia and hyper-responsivity at 1500 ppm, and reduced maternal body weight gain at 1500 during the exposure period only and at 3000 ppm from initiation of exposure to GD20. At Caesarean section on GD20, no adverse effects on implantation, number and viability of fetuses, or fetal sex distribution were observed. Litter weight and mean fetal weight was reduced at 3000 ppm and mean fetal weight was reduced at 1500 ppm. Instances of reduced or unossified skeletal elements occurred at the same dose levels. Mean fetal weight was also reduced at 250 ppm but not at 750 ppm. Extensive statistical analysis of fetal body weight data support the conclusion that there is no toxicologically significant dose-related effect on fetal body weight at or below 750 ppm. Low incidences (≤2.5%) of various malformations occurred in the 250, 1500, and 3000 ppm groups; there was no increase in the incidence of specific or total malformations with increased exposure and thus these were not attributed to toluene.

In this Toluene study, the maternal toxicity NOAEL was 750 ppm with a defined maternal and developmental toxicity LOAEL of 1500 ppm.     

Effects of catalpalactone on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells

The effects of catalpalactone on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Catalpalactone at 5–30 μM decreased intracellular dopamine content with the IC₅₀ value of 22.1 μM. Catalpalactone at 5–20 μM, but not 30 μM, did not alter cell viability. Catalpalactone at 20 μM inhibited tyrosine hydroxylase (TH) and aromatic-l-amino acid decarboxylase (AADC) activities. Catalpalactone also decreased cyclic AMP levels and inhibited TH phosphorylation. In addition, catalpalactone at 20 μM reduced the increases in dopamine levels induced by L-DOPA (20–50 μM). Catalpalactone (5–30 μM) associated with L-DOPA (50–100 μM) enhanced L-DOPA-induced cytotoxicity at 48 h, which was prevented by N-acetyl-l-cysteine. These results suggest that catalpalactone inhibited dopamine biosynthesis by reducing TH and AADC activities and enhanced L-DOPA-induced cytotoxiciy in PC12 cells.