Interaction of rat alveolar macrophages with dental composite dust.

BACKGROUND: Dental composites have become the standard filling material to restore teeth, but during the placement of these restorations, high amounts of respirable composite dust (<5 µm) including many nano-sized particles may be released in the breathing zone of the patient and dental operator. Here we tested the respirable fraction of several composite particles for their cytotoxic effect using an alveolar macrophage model system. ​METHODS: Composite dust was generated following a clinical protocol, and the dust particles were collected under sterile circumstances. Dust was dispersed in fluid, and 5-µm-filtered to enrich the respirable fractions. Quartz DQ12 and corundum were used as positive and negative control, respectively. Four concentrations (22.5 µg/ml, 45 µg/ml, 90 µg/ml and 180 µg/ml) were applied to NR8383 alveolar macrophages. Light and electron microscopy were used for subcellular localization of particles. Culture supernatants were tested for release of lactate dehydrogenase, glucuronidase, TNF-α, and H2O2. RESULTS: Characterization of the suspended particles revealed numerous nano-sized particles but also many high volume particles, most of which could be removed by filtering. Even at the highest concentration (180 µg/ml), cells completely cleared settled particles from the bottom of the culture vessel. Accordingly, a mixture of nano- and micron-scaled particles was observed inside cells where they were confined to phagolysosomes. The filtered particle fractions elicited largely uniform dose-dependent responses, which were elevated compared to the control only at the highest concentration, which equaled a mean cellular dose of 120 pg/cell. A low inflammatory potential was identified due to dose-dependent release of H2O2 and TNF-α. However, compared to the positive control, the released levels of H2O2 and TNF-α were still moderate, but their release profiles depended on the type of composite. CONCLUSIONS: Alveolar macrophages are able to phagocytize respirable composite dust particle inclusive nanoparticles. Since NR8383 cells tolerate a comparatively high cell burden (60 pg/cell) of each of the five materials with minimal signs of cytotoxicity or inflammation, the toxic potential of respirable composite dust seems to be low. These results are reassuring for dental personnel, but more research is needed to characterize the actual exposure and uptake especially of the pure nano fraction.

Opinion of the Scientific Committee on Consumer Safety (SCCS) - Revision of the opinion on the safety of the use of Silica, Hydrated Silica, and Silica Surface Modified with Alkyl Silylates (nano form) in cosmetic products.

CONCLUSION OF THE OPINION: The SCCS has concluded that the evidence, both provided in the submission and that available in scientific literature, is inadequate and insufficient to allow drawing any firm conclusion either for or against the safety of any of the individual SAS material, or any of the SAS categories that are intended for use in cosmetic products. As the SCCS has not been able to conclude on the safety of the synthetic amorphous silica (SAS) materials included in the current submission, the Applicant is advised to follow the SCCS Guidance on Risk Assessment of Nanomaterials (SCCS/1484/12). A brief summary is provided to enable/facilitate future evaluation of the SAS materials in cosmetic products.

Nano-TiO2 modulates the dermal sensitization potency of dinitrochlorobenzene after topical exposure.

BACKGROUND: Little is known about the impact of engineered nanoparticles (ENPs) on skin sensitization caused by chemicals. OBJECTIVES: We determined the ability of different ENPs (TiO2 , Ag and SiO2 ) and aged paint particles containing ENPs to modulate dermal sensitization by a known potent dermal sensitizer. METHODS: The fur of BALB/c mice in the area around the ears was cut with scissors 1 day prior to topical exposure to ENPs (0·4, 4 or 40 mg mL(-1) ), paint particles containing ENPs (4 mg mL(-1) ) or vehicle (day 0). On days 1, 2 and 3, the mice received dermal applications on the back of both ears of 2,4-dinitrochlorobenzene (DNCB) or vehicle. The stimulation index (SI) was calculated on day 6. RESULTS: Topical exposure to TiO2 , Ag or SiO2 ENPs, or aged paint particles followed by vehicle treatment as a control, did not influence the SI. When 4 mg mL(-1) TiO2 ENPs were applied prior to DNCB sensitization, we found an increased SI compared with vehicle-exposed mice prior to DNCB sensitization. Furthermore, an increased titanium concentration was found in the draining lymph node cells of this group. Topical exposure to Ag or SiO2 ENPs or aged paint particles prior to DNCB sensitization did not influence the SI. CONCLUSIONS: We have demonstrated that topical exposure to TiO2 ENPs increases chemical-induced dermal sensitization.

Negative impact of occupational exposure on surgical outcome in patients with rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) is a frequent condition that is treated by functional endoscopic sinus surgery (FESS) when medical treatment fails. Endogenous as well as exogenous factors may be responsible for persisting symptoms after FESS. The role of occupational exposures on success of FESS has never been investigated. METHODS: In this case-control study, we tested the hypothesis that the outcome of FESS procedures is related to exposures at work. Questionnaires were sent to 890 patients who had undergone one or more FESS procedures and to 182 controls. Three independent experts assessed blindly the reported work exposures to inhaled agents. The relationship between exposure and the number of FESS procedures was analyzed. RESULTS: Relevant occupational exposure was reported by 25% of all responding patients undergoing FESS (n = 467) and 12% of controls (n = 69). The prevalence of occupational exposures increased linearly with the number of FESS procedures from 21% in those who had one FESS to 44% in those who had four or more FESS (χ(2)  = 12.74, P < 0.001). Logistic regression analysis with adjustments for potential confounders, including smoking, atopy, and asthma, confirmed that the odds ratio (OR) for reporting occupational exposures was significantly higher in those needing more than one FESS (OR = 1.64) or more than two FESS (OR = 1.97). These results were mainly driven by exposure to low molecular weight agents. CONCLUSION: Exposure at work appears to be a risk factor for the occurrence of CRS and for its recurrence or persistence, as evidenced by the need for revision surgery.

Worrying exposure to trace elements in the population of Kinshasa, Democratic Republic of Congo (DRC).

BACKGROUND AND OBJECTIVES: The particularly high rate of urbanization in Kinshasa (Democratic Republic of Congo) is associated with environmental degradation. Outdoor and indoor air pollution, as well as water pollution and waste accumulation, are issues of major concern. However, little documented information exists on the nature and extent of this pollution. A biomonitoring study was conducted to document exposure to trace elements in a representative sample of the population in Kinshasa. METHODS: Fifteen trace elements were measured by ICP-MS, CV-AAS, or HG-AFS in spot urine samples from 220 individuals (50.5% women) aged 6-70 years living in the urban area and from 50 additional subjects from the rural area of Kinshasa. Data were compiled as geometric means and selected percentiles, expressed without (µg/L) or with creatinine adjustment (µg/g cr). RESULTS: Overall, living in urban Kinshasa was associated with elevated levels of several parameters in urine as compared to the population living in the rural area (Asi, Ba, Cd, Cr, and V) as well as compared to an urban population of the southeast of Congo (Al, As, Cd, Cr, Cu, Pb, Mn, Ni, Se, V, and Zn). Elevated levels were also found by comparison with the reference values in databases involving American, Canadian, French, or German populations. CONCLUSIONS: This study provides the first biomonitoring database in the population of Kinshasa, revealing elevated levels for most urinary TE as compared to other databases. Toxicologically relevant elements such as Al, As, Cd, Pb, and Hg reach levels of public health concern.

The miniaturized enzyme-modified comet assay for genotoxicity testing of nanomaterials.

The in vitro comet assay is a widely applied method for investigating genotoxicity of chemicals including engineered nanomaterials (NMs). A big challenge in hazard assessment of NMs is possible interference between the NMs and reagents or read-out of the test assay, leading to a risk of biased results. Here, we describe both the standard alkaline version of the in vitro comet assay with 12 mini-gels per slide for detection of DNA strand breaks and the enzyme-modified version that allows detection of oxidized DNA bases by applying lesion-specific endonucleases (e.g., formamidopyrimidine DNA glycosylase or endonuclease III). We highlight critical points that need to be taken into consideration when assessing the genotoxicity of NMs, as well as basic methodological considerations, such as the importance of carrying out physicochemical characterization of the NMs and investigating uptake and cytotoxicity. Also, experimental design-including treatment conditions, cell number, cell culture, format and volume of medium on the plate-is crucial and can have an impact on the results, especially when testing NMs. Toxicity of NMs depends upon physicochemical properties that change depending on the environment. To facilitate testing of numerous NMs with distinct modifications, the higher throughput miniaturized version of the comet assay is essential.

Lung exposure to nanoparticles modulates an asthmatic response in a mouse model.

The aim of this study was to investigate the modulation of an asthmatic response by titanium dioxide (TiO2) or gold (Au) nanoparticles (NPs) in a murine model of diisocyanate-induced asthma. On days 1 and 8, BALB/c mice received 0.3% toluene diisocyanate (TDI) or the vehicle acetone-olive oil (AOO) on the dorsum of both ears (20 µL). On day 14, the mice were oropharyngeally dosed with 40 µL of a NP suspension (0.4 mg·mL⁻¹ (∼0.8 mg·kg⁻¹) TiO2 or Au). 1 day later (day 15), the mice received an oropharyngeal challenge with 0.01% TDI (20 µL). On day 16, airway hyperreactivity (AHR), bronchoalveolar lavage (BAL) cell and cytokine analysis, lung histology, and total serum immunoglobulin E were assessed. NP exposure in sensitised mice led to a two- (TiO2) or three-fold (Au) increase in AHR, and a three- (TiO2) or five-fold (Au) increase in BAL total cell counts, mainly comprising neutrophils and macrophages. The NPs taken up by BAL macrophages were identified by energy dispersive X-ray spectroscopy. Histological analysis revealed increased oedema, epithelial damage and inflammation. In conclusion, these results show that a low, intrapulmonary doses of TiO2 or Au NPs can aggravate pulmonary inflammation and AHR in a mouse model of diisocyanate-induced asthma.

Immunological determinants in a mouse model of chemical-induced asthma after multiple exposures.

In a mouse model of chemical-induced asthma, we investigated the effects of multiple challenges, using toluene diisocyanate (TDI), a known cause of occupational asthma. On days 1 and 7, BALB/c mice received TDI or vehicle (acetone/olive oil). On days 10, 13 and 16 the mice received an intranasal instillation of TDI. Ventilatory function (Penh) was monitored by whole body plethysmography for 40 min after each challenge. Reactivity to methacholine was measured 22 h later. Pulmonary inflammation, TNF-alpha and MIP-2 levels were assessed 24 h after the last challenge by broncho-alveolar lavage (BAL). Other immunological parameters included total IgE, lymphocyte sub-populations in auricular and cervical lymph nodes, and IL-4, IFN-gamma and IL-13 levels in supernatants of lymph node cells, cultured with or without concanavalin A. Early ventilatory function and airway reactivity increased in all groups that received a dermal application and one or multiple intranasal challenges of TDI. After multiple challenges, lung inflammation was characterized by neutrophils (approximately 15%), and eosinophils (approximately 4%), along with an increase in BAL MIP-2 and TNF-alpha levels. The auricular and cervical lymph node cells of all sensitized mice showed an increase in B cells, Th cells and an increased concentration of in vitro release of IL-4, IFN-gamma and IL-13 after stimulation with concanavalin A. Total serum IgE was elevated in dermally TDI-sensitized mice. This protocol including multiple challenges results in a model that resembles human asthma, indicating that responses found in the model using a single challenge could be a good first indication for the development of asthma.

Co-cultures of multiple cell types mimic pulmonary cell communication in response to urban PM10.

The current authors evaluated whether a system of co-cultures of relevant cells (pneumocytes (A549), macrophages (THP-1), mast cells (HMC-1) and endothelial cells (EAHY926)) would mimic the responses to particles with a 50% cut-off aerodynamic diameter of 10 microm (PM(10)) previously reported in vivo. The role of mast cells was considered of special interest. Single cultures, bicultures (A549 + HMC-1 in a 10:1 ratio; THP-1 + HMC-1 in a 2:1 ratio) and tricultures (A549 + THP-1 + HMC-1 in a 10:2:1 ratio) were exposed to urban PM(10) (24 h at 0, 10, 30 or 100 microg x cm(-2)). Additionally, EAHY926 cells were introduced in inserts above the tricultures. The released cytokines were evaluated with a fluorescence-activated cell sorter array system. THP-1 + HMC-1 bicultures and the tricultures released more granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1beta, interleukin (IL)-1beta, IL-8, IL-6, tumour necrosis factor-alpha and MIP-1alpha in response to PM(10) than the sum of the single cultures. Tricultures with EAHY926 released more G-CSF, MIP-1alpha, IL-8 and MIP-1beta than the EAHY926 single culture. The bicultures, tricultures and tricultures with EAHY926 provide results that are consistent with the local and systemic effects previously described for particulate matter effects, i.e. inflammation, endothelial dysfunction and bone marrow cell mobilisation. Mast cells seem to play a significant role in the co-culture responses.

Enhanced peripheral thrombogenicity after lung inflammation is mediated by platelet-leukocyte activation: role of P-selectin.

BACKGROUND: Inhaled ultrafine particles trigger peripheral thrombotic complications. METHODS: We have analyzed the systemic prothrombotic risk following lung inflammation induced by pulmonary carbon nanotubes (CNTs). RESULTS: Intratracheal instillation in Swiss mice of 200 and 400 microg of multiwall ground CNTs triggered substantial lung neutrophil, but not macrophage influx, 24 h later. The detection of circulating platelet-leukocyte conjugates exclusively 6 h after CNT instillation pointed to early but transient activation of circulating platelets. At 24 h, elevated plasma procoagulant microvesicular tissue factor activity was found in CNT-exposed but not in saline-exposed mice. However, at 24 h, both the tail and jugular vein bleeding times were prolonged in CNT-exposed but not in saline-exposed mice, arguing against strong CNT-induced platelet activation at this point. Nevertheless, at 24 h, enhanced peripheral thrombogenicity was detected in CNT-exposed but not in saline-exposed mice, via quantitative photochemically induced carotid artery thrombosis measurements. P-selectin neutralization abrogated platelet-leukocyte conjugate formation and microvesicular tissue factor generation, and abolished the CNT-induced thrombogenicity amplification. In contrast, the weak vascular injury-triggered thrombus formation in saline-treated mice was not affected by P-selectin neutralization at 24 h. CONCLUSIONS: The mild CNT-induced lung inflammation translates via rapid but mild and transient activation of platelets into P-selectin-mediated systemic inflammation. Leukocyte activation leads to tissue factor release, in turn eliciting inflammation-induced procoagulant activity and an associated prothrombotic risk.

Stronger associations between daily mortality and fine particulate air pollution in summer than in winter: evidence from a heavily polluted region in western Europe.

BACKGROUND: Numerous studies have shown a strong association between daily mortality and small particulate with a diameter of <10 microm (PM10) air pollution, but the effects of season have not always been well characterised. AIM: To study the shape of the association between short-term mortality and PM10 across seasons and quintiles of outdoor temperature. DESIGN, SETTING AND PARTICIPANTS: Daily data on mortality (n = 354 357), outdoor temperature and PM10 in Flanders, Belgium, from January 1997 to December 2003, were analysed across warm versus cold periods of the year (April-September v October-March), with seasons and quintiles of outdoor temperature as possible effect modifiers. RESULTS: There was a significant (p<0.001) interaction between PM10 and period of the year in relation to mortality. To allow for non-linearity, daily mean PM10 concentrations were categorised into quartiles. Season-specific PM10 quartiles showed a strong and steep linear association between mortality and PM10 in summer and a less linear association in spring and autumn, whereas in winter the association was less strong and mortality was only increased in the highest PM10 quartile. The effect sizes expressed as the percentage increase in mortality on days in the highest season-specific PM(10) quartile versus the lowest season-specific PM10 quartile were 7.8% (95% CI 6.1 to 9.6) in summer, 6.3% (4.7 to 7.8) in spring, 2.2% (0.58 to 3.8) in autumn and 1.4% (0.06 to 2.9) in winter. An analysis by quintiles of temperature confirmed these effect sizes. CONCLUSION: The short-term effect of particulate air pollution on mortality strongly depends on outdoor temperature, even in a temperate climate.

Optimisation of culture conditions to develop an in vitro pulmonary permeability model.

An in vitro model to study pulmonary translocation was created, using the human cell line Calu-3 and primary rat type II pneumocytes. Cells were seeded on permeable membranes with a 0.4 microm or 3 microm pore size, utilizing different culture conditions such as medium formulation and cell density. The integrity of the cell monolayer was verified by measuring the transepithelial electrical resistance (TEER) and passage of sodium fluorescein. When seeded on inserts with 0.4 microm pore size, the Calu-3 cells and primary rat type II pneumocytes created high TEER values of 949+/-182 Omega cm(2) and 400+/-257 Omega cm(2), respectively. On membranes with 3 microm pores, Calu-3 cells achieved a high TEER value of 500+/-95 Omega cm(2). Our experiments indicate that the culture medium was more critical than the cell density, regarding the influence on TEER values. For both cell types a reduction of serum in the medium resulted in a decrease in TEER value. We established a good ('tight') monolayer of primary type II pneumocytes in Waymouth medium at a cell density of 0.9x10(6) cells/cm(2); the Calu-3 cells should be grown in DMEM medium containing Hepes at 0.75x10(6) cells/cm(2).

Release of monomers from composite dust.

OBJECTIVES: Dental personnel are more at risk to develop asthmatic disease, but the exact reason is so far unknown. During abrasive procedures, dental personnel are exposed to nano-sized dust particles released from dental composite. The aim of this study was to investigate whether respirable composite dust may also release monomers. METHODS: Respirable (<5µm) composite dust was collected and the release of methacrylate monomers and Bisphenol A (BPA) in water and ethanol was evaluated by liquid chromatography/mass spectroscopy (LC-MS/MS). The dust was ultra-morphologically and chemically analyzed by transmission electron microscopy and energy-dispersive X-ray spectroscopy (TEM-EDS). RESULTS: LC-MS/MS analysis revealed that, irrespective of the type of composite, the respirable fraction of composite dust may release relatively high concentrations of unpolymerized methacrylate monomers, both in water and ethanol. Higher release was observed in ethanol. The endocrine disruptor BPA also emanated from the composite dust particles. TEM showed that most particles were nano-sized, although particle size ranged between 6nm and 5µm with a mode value between 12 and 39nm. Most particles consisted of several filler particles in resin matrix, although single nano-filler particles could also be observed. Elemental analysis by TEM-EDS proved that the particles collected on the filters originated from the dental composites. CONCLUSION: Theoretically, composite dust may function as a vehicle to transport monomers deeply into the respiratory system. The results of this study may shed another light on the increasing incidence of respiratory disease among dental personnel, and more care should be taken to prevent inhalation of composite dust. CLINICAL SIGNIFICANCE: Special care should be taken to prevent inhalation of composite dust, as the dust particles may release methacrylate monomers.

In vitro study of the pulmonary translocation of nanoparticles: a preliminary study.

Recent studies indicate that inhaled ultrafine particles can pass into the circulation. To study this translocation in an in vitro model three types of pulmonary epithelial cells were examined. The integrity of the cell monolayer was verified by measuring the transepithelial electrical resistance (TEER) and passage of sodium fluorescein. TEER was too low in A549 cells. In these preliminary experiments, TEER values of 1007+/-300 and 348+/-62 Omega cm2 were reached for the Calu-3 cell line, using permeable membranes of 0.4 and 3 microm pore size, respectively. Growing primary rat type II pneumocytes on 0.4 microm pores, a TEER value of 241+/-90 Omega cm2 was reached on day 5; on 3 microm pores, no acceptable high TEER value was obtained. Translocation studies were done using 46 nm fluorescent polystyrene particles. When incubating polystyrene particles on membranes without a cellular monolayer, significant translocation was only observed using 3 microm pores: 67.5% and 52.7% for carboxyl- and amine-modified particles, respectively. Only the Calu-3 cell line was used in an initial experiment to investigate the translocation: on 0.4 microm pores no translocation was observed, on 3 microm pores approximately 6% translocation was observed both for carboxyl- and amine-modified particles.

Death and cell cycle progression are differently conditioned by the AgNP size in osteoblast-like cells.

Silver nanoparticles (AgNPs) are useful to a wide range of consumer's and medical products, due to their antimicrobial and anti-inflammatory activities. AgNPs have been used to prevent the microbial colonization, therefore decreasing the risk of infection, on implantable devices, tumor prostheses, bone cement and surgical instruments. However, the putative toxicity of AgNPs to bone cells is still poorly understood. Therefore, this study aimed to contribute to enlighten the role of ionic silver release of small sized NPs on the biological outcomes of bone cells, in particular to what concerns to induction of cytotoxic and genotoxic effects. To achieve that goal osteoblast-like MG-63 cells were exposed to well characterized PVP coated AgNPs of two different primary sizes (10nm and 20nm) and evaluated after 24 and 48h. Our results showed that, the smaller sized AgNPs (10nm) are more reactive and prone to form large aggregates, being therefore mandatory to provide a careful characterization of the particles, before the toxicity assessment. We also demonstrate that for short period exposures (up to 48h) ionic silver (from AgNO3) is more toxic than the corresponding dose of AgNP. However, when assessing longer term exposures by the clonogenic assay, we demonstrated the inverse effect, the AgNPs turn out being more toxic, completely inhibiting plate efficiency. Therefore, AgNPs toxicity cannot be attributed to the dissociated Ag+ alone. Also, when comparing size-dependent effects, we demonstrate that AgNP20 were found to induce a cell cycle arrest at G0/G1 and apoptosis, while AgNP10 did not induce a cytostatic effect, but rather induced necrosis. Finally, combining the chemical and toxicological profiles of both AgNP sizes, we hypothesize that the size dependent AgNP toxicity may be associated in part with the NPs interference with the cell membranes and consequent uptake/adsorption processes.

Passage of inhaled particles into the blood circulation in humans.

BACKGROUND: Pollution by particulates has been consistently associated with increased cardiovascular morbidity and mortality. However, the mechanisms responsible for these effects are not well-elucidated. METHODS AND RESULTS: To assess to what extent and how rapidly inhaled pollutant particles pass into the systemic circulation, we measured, in 5 healthy volunteers, the distribution of radioactivity after the inhalation of "Technegas," an aerosol consisting mainly of ultrafine (99m)Technetium-labeled carbon particles (<100 nm). Radioactivity was detected in blood already at 1 minute, reached a maximum between 10 and 20 minutes, and remained at this level up to 60 minutes. Thin layer chromatography of blood showed that in addition to a species corresponding to oxidized (99m)Tc, ie, pertechnetate, there was also a species corresponding to particle-bound (99m)Tc. Gamma camera images showed substantial radioactivity over the liver and other areas of the body. CONCLUSIONS: We conclude that inhaled (99m)Tc-labeled ultrafine carbon particles pass rapidly into the systemic circulation, and this process could account for the well-established, but poorly understood, extrapulmonary effects of air pollution.

Putrescine uptake in rat type II pneumocytes correlates with gamma-glutamyltransferase activity.

gamma-Glutamyltransferase (gamma GT) is a key enzyme in glutathione metabolism and it is thought also to play a role in the uptake of polyamines such as putrescine. The aim of our study was to investigate if changes in gamma GT activity would alter total putrescine uptake [P(up)(tot)], as well as more specific uptake via the gamma GT pathway [P(up)(gamma GT)]. Forty-eight hours after their isolation, rat type II cells were exposed to 30, 60 or 125 microM L-buthionine-[SR]-sulfoximine (BSO) for 3 hr; 200 or 800 microM tertiary-butylhydroperoxide (t-BOOH) for 40 min; 10, 100 or 1000 microM paraquat (PQ) for 1 hr; and 60 or 85% O2 for 48 hr. The gamma GT activity, P(up)(tot) and P(up)(gamma GT) (assessed by inhibiting gamma GT) were measured immediately after the exposure to hyperoxia, or 24 hr after treatment with BSO, t-BOOH or PQ. From previous studies, it is known that these experimental conditions increased (BSO, 200 microM t-BOOH) or decreased (800 microM t-BOOH, PQ, hyperoxia) gamma GT activity. There was a strong correlation between the changes in gamma GT activity and the changes in P(up)(gamma GT) (r = 0.81, p < 0.001). These findings support the hypothesis that gamma GT partly regulates the uptake of putrescine, one of the polyamines required for cell growth and differentiation.

Increase in gamma-glutamyltransferase by glutathione depletion in rat type II pneumocytes.

The purpose of our study was to investigate the effect of oxidative stress or intracellular glutathione (GSH) depletion on gamma-glutamyltransferase (gamma-GT) activity in cultured type II pneumocytes. Twenty-four hours after isolation, primary cultures of rat type II pneumocytes were preincubated with one of four compounds: 15, 30, 60, 125, 250 microM L-buthionine-[SR]-sulfoximine (BSO) for 3 h; 100, 200, 400, 800 microM tertiary-butylhydroperoxide (t-BOOH) for 45 min; 10, 25, 50, 100 microM menadione for 15 min; 100, 1000 microM paraquat for 1 h. GSH levels, H2O2 and O2.- generation were measured immediately after the incubation, gamma-GT activity and GSH levels also up to 24 h or 48 h later. Exposure to BSO led to a persistent GSH depletion without increase in H2O2 or O2.- production, together with a dose and time-dependent increase (doubling) of gamma-GT activity with a nonsignificant increase in gamma-GT mRNA expression 24 h after exposure to BSO. Exposure to 100 microM menadione, which increased H2O2 production, decreased gamma-GT activity. t-BOOH or paraquat did not give rise to a measurable increase in H2O2 or O2.-. Paraquat did not affect initial GSH levels, but increased GSH and decreased gamma-GT activity 24 h later. t-BOOH (400 and 800 microM) initially decreased GSH, and tended to increase GSH 24 h later, 100 and 200 microM increased gamma-GT activity 24 h later, but 800 microM decreased it. Restoration of intracellular GSH levels by addition of GSH to the culture medium completely prevented the increase in gamma-GT activity by BSO, while the addition of catalase or DMTU had no effect. We conclude that at least two effects are operating upon gamma-GT activity: GSH depletion seems to increase gamma-GT activity, while exposure to compounds generating oxidative stress correlates with a decrease in gamma-GT activity.

Expression in Bacillus subtilis of the glycosomal glyceraldehyde-phosphate dehydrogenase gene from Trypanosoma brucei.

The cloned T brucei GAPDH gene was inserted within the B subtilis GAPDH gene, carried by pUC18. Upon transformation of B subtilis by this plasmid, not able to replicate in this host, the whole plasmid was inserted in the resident chromosome, presumably by a single recombination event between homologous, chromosomal and plasmid-borne sequences. The heterologous gene was expressed, as revealed by immunological reaction with monoclonal antibodies, recognizing specifically T brucei GAPDH. T brucei GAPDH, having little or no enzyme activity, comprises about 1.56% of cellular proteins. Peptide mapping showed that a fusion of a 7.5-kDa peptide had occurred to the N-terminal part of T brucei GAPDH. This fused protein is presumably the N-terminal part of B subtilis GAPDH, in agreement with the construction of the integrative plasmid.

Paracetamol (acetaminophen) cytotoxicity in rat type II pneumocytes and alveolar macrophages in vitro.

Paracetamol (acetaminophen, APAP) liver and kidney cytotoxicity is associated with bioactivation by P450 and/or prostaglandin H synthetase (PGHS) to a reactive metabolite, which depletes GSH, covalently binds to proteins, and leads to oxidative stress. Although APAP may also damage the lung, little is known about the mechanism by which this occurs. We studied the in vitro 24-hr-old type II pneumocytes. A time- and concentration-dependent decrease in intracellular GSH occurred in freshly isolated type II pneumocytes and alveolar macrophages exposed to subtoxic (