Asparagine transport through SLC1A5/ASCT2 and SLC38A5/SNAT5 is essential for BCP-ALL cell survival and a potential therapeutic target.

B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) blasts strictly depend on the transport of extra-cellular asparagine (Asn), yielding a rationale for L-asparaginase (ASNase) therapy. However, the carriers used by ALL blasts for Asn transport have not been identified yet. Exploiting RS4;11 cells as BCP-ALL model, we have found that cell Asn is lowered by either silencing or inhibition of the transporters ASCT2 or SNAT5. The inhibitors V-9302 (for ASCT2) and GluγHA (for SNAT5) markedly lower cell proliferation and, when used together, suppress mTOR activity, induce autophagy and cause a severe nutritional stress, leading to a proliferative arrest and a massive cell death in both the ASNase-sensitive RS4;11 cells and the relatively ASNase-insensitive NALM-6 cells. The cytotoxic effect is not prevented by coculturing leukaemic cells with primary mesenchymal stromal cells. Leukaemic blasts of paediatric ALL patients express ASCT2 and SNAT5 at diagnosis and undergo marked cytotoxicity when exposed to the inhibitors. ASCT2 expression is positively correlated with the minimal residual disease at the end of the induction therapy. In conclusion, ASCT2 and SNAT5 are the carriers exploited by ALL cells to transport Asn, and ASCT2 expression is associated with a lower therapeutic response. ASCT2 may thus represent a novel therapeutic target in BCP-ALL.

ALL blasts drive primary mesenchymal stromal cells to increase asparagine availability during asparaginase treatment.

Mechanisms underlying the resistance of acute lymphoblastic leukemia (ALL) blasts to l-asparaginase are still incompletely known. Here we demonstrate that human primary bone marrow mesenchymal stromal cells (MSCs) successfully adapt to l-asparaginase and markedly protect leukemic blasts from the enzyme-dependent cytotoxicity through an amino acid trade-off. ALL blasts synthesize and secrete glutamine, thus increasing extracellular glutamine availability for stromal cells. In turn, MSCs use glutamine, either synthesized through glutamine synthetase (GS) or imported, to produce asparagine, which is then extruded to sustain asparagine-auxotroph leukemic cells. GS inhibition prevents mesenchymal cells adaptation to l-asparaginase, lowers glutamine secretion by ALL blasts, and markedly hinders the protection exerted by MSCs on leukemic cells. The pro-survival amino acid exchange is hindered by the inhibition or silencing of the asparagine efflux transporter SNAT5, which is induced in mesenchymal cells by ALL blasts. Consistently, primary MSCs from ALL patients express higher levels of SNAT5 (P < .05), secrete more asparagine (P < .05), and protect leukemic blasts (P < .05) better than MSCs isolated from healthy donors. In conclusion, ALL blasts arrange a pro-leukemic amino acid trade-off with bone marrow mesenchymal cells, which depends on GS and SNAT5 and promotes leukemic cell survival during l-asparaginase treatment.

Myeloma Cells Deplete Bone Marrow Glutamine and Inhibit Osteoblast Differentiation Limiting Asparagine Availability.

Multiple myeloma (MM) cells consume huge amounts of glutamine and, as a consequence, the amino acid concentration is lower-than-normal in the bone marrow (BM) of MM patients. Here we show that MM-dependent glutamine depletion induces glutamine synthetase in stromal cells, as demonstrated in BM biopsies of MM patients, and reproduced in vitro by co-culturing human mesenchymal stromal cells (MSCs) with MM cells. Moreover, glutamine depletion hinders osteoblast differentiation of MSCs, which is also severely blunted by the spent, low-glutamine medium of MM cells, and rescued by glutamine restitution. Glutaminase and the concentrative glutamine transporter SNAT2 are induced during osteoblastogenesis in vivo and in vitro, and both needed for MSCs differentiation, pointing to enhanced the requirement for the amino acid. Osteoblastogenesis also triggers the induction of glutamine-dependent asparagine synthetase (ASNS), and, among non-essential amino acids, asparagine rescues differentiation of glutamine-starved MSCs, by restoring the transcriptional profiles of differentiating MSCs altered by glutamine starvation. Thus, reduced asparagine availability provides a mechanistic link between MM-dependent Gln depletion in BM and impairment of osteoblast differentiation. Inhibition of Gln metabolism in MM cells and supplementation of asparagine to stromal cells may, therefore, constitute novel approaches to prevent osteolytic lesions in MM.

Urinary biomarkers of nucleic acid oxidation and methylation in workers exposed to low concentrations of benzene.

The study aims to investigate the influence of exposure to low concentrations of benzene on urinary biomarkers of nucleic acid oxidative damage and methylation. Benzene exposure was characterized for 93 coke production workers by measuring both airborne benzene and S-phenylmercapturic acid (SPMA) and unmodified benzene (U-B) in urine samples, collected at the end of the shift (ES) and at the next morning before shift (next BS). In the same urinary samples, biomarkers of nucleic acid oxidative damage and methylation were determined. Urinary concentrations of cotinine and creatinine were also determined to evaluate the smoking effect and to normalize urinary concentrations of analytes, respectively. The biomarkers of benzene internal dose, of oxidative damage (8-hydroxyy-7,8-dihydroguanine, 8-hydroxy-7,8-dihydroguanosine and 8-hydroxy-7,8-2'deoxyguanosine) and some of the biomarkers of nucleic acid methylation (5-Methyl-Cytosine, 1-Methyl-Guanine and 7-Methyl-Guanine) were higher in the ES than the next BS samples. Positive associations between ES 5-Methyl-Cytosine and both SPMA and U-B were found. In conclusion, occupational exposure to low levels of benzene seems to be related to urinary ES 5-Methyl-Cytosine that could be a possible biomarker to evaluate the changes of the nucleic acid methylation status.

Idiopathic pulmonary fibrosis and occupational risk factors.

Idiopathic pulmonary fibrosis (IPF) is a rare lung disease of unknown origin that rapidly leads to death. However, the rate of disease progression varies from one individual to another and is still difficult to predict. The prognosis of IPF is poor, with a median survival of three to five years after diagnosis, without curative therapies other than lung transplantation. The factors leading to disease onset and progression are not yet completely known. The current disease paradigm is that sustained alveolar epithelial micro-injury caused by environmental triggers (e.g., cigarette smoke, microaspiration of gastric content, particulate dust, viral infections or lung microbial composition) leads to alveolar damage resulting in fibrosis in genetically susceptible individuals. Numerous epidemiological studies and case reports have shown that occupational factors contribute to the risk of developing IPF. In this perspective, we briefly review the current understanding of the pathophysiology of IPF and the importance of occupational factors in the pathogenesis and prognosis of the disease. Prompt identification and elimination of occult exposure may represent a novel treatment approach in patients with IPF.

Sulphurous thermal water inhalation impacts respiratory metabolic parameters in heavy smokers.

Sulphurous thermal water inhalations have been traditionally used in the treatment of airway diseases. In vivo and in vitro studies reported that they ameliorate mucus rheology, mucociliary clearance and reduce inflammation. Cigarette smoking induces an inflammatory damage, with consequent remodeling of respiratory airways, which in turn affect pulmonary functions. Despite the anti-inflammatory effects of H2S are clinically documented in several airway inflammatory diseases, data on the effects of sulphurous thermal water treatment on pulmonary function and biomarkers of airways inflammation in smokers are still scant. Therefore, we investigated whether a conventional cycle of sulphurous thermal water inhalation produced changes in markers of respiratory inflammation and function. A cohort of 504 heavy current and former smokers underwent 10-day cycles of sulphurous thermal water inhalation. Pulmonary function and metabolic analyses on exhaled breath condensate were then performed at day 0 and after the 10-day treatment. Spirometric data did not change after spa therapy, while exhaled breath condensate analysis revealed that a single 10-day cycle of sulphurous water inhalation was sufficient to induce a statistically significant increase of citrulline levels along with a decrease in ornithine levels, thus shifting arginine metabolism towards a reduced nitric oxide production, i.e. an anti-inflammatory profile. Overall, sulphurous thermal water inhalation impacts on arginine catatabolic intermediates of airways cells, shifting their metabolic balance towards a reduction of the inflammatory activity, with potential benefits for smokers.

Determination of synthetic and natural cannabinoids in oral fluid by solid-phase microextraction coupled to gas chromatography/mass spectrometry: A pilot study.

In 2008, several synthetic cannabinoids were detected in herbal smoking blends sold on websites and in the so-called "smart shops". These compounds, as well as new psychoactive substances, flooded the market of illicit drugs and are sold at street level. Development and validation of rapid analytical methods for the detection and quantification of synthetic cannabinoids in biological matrices are essential for the investigation of pharmacokinetic, pharmacodynamic and toxicological properties. In this pilot study, the potential of direct immersion solid-phase microextraction coupled to GC/MS for the determination of synthetic and natural cannabinoids in oral fluid was proved. Validation proved the suitability of the developed method for the determination of the analytes at trace levels in oral fluid: linearity was assessed in the LOQ - 1000 ng/mL range, whereas a good precision was observed with CV below 20%. The method was then applied to more than 100 real oral fluid samples collected from a population of young students, showing a positivity rate of 3.8%.

7-Hydroxymatairesinol improves body weight, fat and sugar metabolism in C57BJ/6 mice on a high-fat diet.

7-Hydroxymatairesinol (7-HMR) is a plant lignan abundant in various concentrations in plant foods. The objective of this study was to test HMRLignan™, a purified form of 7-HMR, and the corresponding Picea abies extract (total extract P. abies; TEP) as dietary supplements on a background of a high-fat diet (HFD)-induced metabolic syndrome in mice and in the 3T3-L1 adipogenesis model. Mice, 3 weeks old, were fed a HFD for 60 d. Subgroups were treated with 3 mg/kg body weight 7-HMR (HMRLignan™) or 10 mg/kg body weight TEP by oral administration. 7-HMR and TEP limited the increase in body weight (-11 and -13 %) and fat mass (-11 and -18 %) in the HFD-fed mice. Epididymal adipocytes were 19 and -12 % smaller and the liver was less steatotic (-62 and -65 %). Serum lipids decreased in TEP-treated mice (-11 % cholesterol, -23 % LDL and -15 % TAG) and sugar metabolism was ameliorated by both lignan preparations, as shown by a more than 70 % decrease in insulin secretion and insulin resistance. The expression of several metabolic genes was modulated by the HFD with an effect that was reversed by lignan. In 3T3-L1 cells, the 7-HMR metabolites enterolactone (ENL) and enterodiol (END) showed a 40 % inhibition of cell differentiation accompanied by the inhibited expression of the adipogenic genes PPARγ, C/EBPα and aP2. Furthermore, END and ENL caused a 10 % reduction in TAG uptake in HEPA 1-6 hepatoma cells. In conclusion, 7-HMR and TEP reduce metabolic imbalances typical of the metabolic syndrome and obesity in male mice, whereas their metabolites inhibit adipogenesis and lipid uptake in vitro.

Biomarkers of exposure to stainless steel tungsten inert gas welding fumes and the effect of exposure on exhaled breath condensate.

The respiratory tract is the main target organ of the inhaled hexavalent chromium (Cr-VI) and nickel (Ni) contained in stainless steel (SS) welding fumes (WFs). The aim of this study was to investigate the Cr and Ni content of the exhaled breath condensate (EBC) of SS tungsten inert gas (TIG) welders, and relate their concentrations with oxidative stress and inflammatory biomarkers. EBC and urine from 100 SS TIG welders were collected pre-(T0) and post-shift (T1) on a Friday, and pre-shift (T2) on the following Monday morning. Both EBC and urinary Cr concentrations were higher at T1 (0.08 µg/L and 0.71 µg/g creatinine) and T0 (0.06 µg/L and 0.74 µg/g creatinine) than at T2 (below the limit of detection [LOD] and 0.59 µg/g creatinine), and EBC Ni concentrations generally remained

Oligodendroglioma Cells Lack Glutamine Synthetase and Are Auxotrophic for Glutamine, but Do not Depend on Glutamine Anaplerosis for Growth.

In cells derived from several types of cancer, a transcriptional program drives high consumption of glutamine (Gln), which is used for anaplerosis, leading to a metabolic addiction for the amino acid. Low or absent expression of Glutamine Synthetase (GS), the only enzyme that catalyzes de novo Gln synthesis, has been considered a marker of Gln-addicted cancers. In this study, two human cell lines derived from brain tumors with oligodendroglioma features, HOG and Hs683, have been shown to be GS-negative. Viability of both lines depends from extracellular Gln with EC50 of 0.175 ± 0.056 mM (Hs683) and 0.086 ± 0.043 mM (HOG), thus suggesting that small amounts of extracellular Gln are sufficient for OD cell growth. Gln starvation does not significantly affect the cell content of anaplerotic substrates, which, consistently, are not able to rescue cell growth, but causes hindrance of the Wnt/ß-catenin pathway and protein synthesis attenuation, which is mitigated by transient GS expression. Gln transport inhibitors cause partial depletion of intracellular Gln and cell growth inhibition, but do not lower cell viability. Therefore, GS-negative human oligodendroglioma cells are Gln-auxotrophic but do not use the amino acid for anaplerosis and, hence, are not Gln addicted, exhibiting only limited Gln requirements for survival and growth.

Reference Intervals for Urinary Cotinine Levels and the Influence of Sampling Time and Other Predictors on Its Excretion Among Italian Schoolchildren.

(1) Background: Environmental Tobacco Smoke (ETS) exposure remains a public health problem worldwide. The aims are to establish urinary (u-) cotinine reference values for healthy Italian children, to evaluate the role of the sampling time and of other factors on children's u-cotinine excretion. (2) Methods: A cross-sectional study was performed on 330 children. Information on participants was gathered by a questionnaire and u-cotinine was determined in two samples for each child, collected during the evening and the next morning. (3) Results: Reference intervals (as the 2.5th and 97.5th percentiles of the distribution) in evening and morning samples were respectively equal to 0.98⁻4.29 and 0.91⁻4.50 µg L-1 (ETS unexposed) and 1.39⁻16.34 and 1.49⁻20.95 µg L-1 (ETS exposed). No statistical differences were recovered between median values found in evening and morning samples, both in ETS unexposed and exposed. Significant predictors of u-cotinine excretions were ponderal status according to body mass index of children (ß = 0.202; p-value = 0.041 for evening samples; ß = 0.169; p-value = 0.039 for morning samples) and paternal educational level (ß = -0.258; p-value = 0.010; for evening samples; ß = -0.013; p-value = 0.003 for morning samples). (4) Conclusions: The results evidenced the need of further studies for assessing the role of confounding factors on ETS exposure, and the necessity of educational interventions on smokers for rising their awareness about ETS.

Biological monitoring of exposure to low concentrations of benzene in workers at a metallurgical coke production plant: new insights into S-phenylmercapturic acid and urinary benzene.

CONTEXT: Urinary S-phenylmercapturic acid (SPMA) and benzene (U-Ben) are usually measured at the end of the work shift (ES), although their kinetic of elimination is not clearly known. OBJECTIVE: To investigate SPMA and U-Ben elimination 16 h after the ES, in 93 coke production workers exposed to low benzene concentrations. MATERIALS AND METHODS: Airborne benzene (A-Ben) was measured by passive samplings, while SPMA, U-Ben, methyl-tert-butyl ether (U-MTBE), cotinine (U-Cot) and creatinine were determined on urine samples collected at ES and before the beginning of the next work shift (next BS). RESULTS: Median A-Ben concentrations were 17.2 µg/m3 in the personal and 34.7 µg/m3 in the stationary samplings. SPMA was always detectable, whereas U-Ben was below the limit of quantification in 26.7% of the ES and 35.6% of the next BS samples, and U-MTBE in more than the 80.0% of the samples. At both the sampling times, SPMA and U-Ben showed a positive dependence on personal A-Ben, as well as on creatinine and U-Cot values. DISCUSSION AND CONCLUSION: SPMA and U-Ben at the next BS were dependent on the exposure to low benzene concentrations suffered in the previous work shift, prompting a reconsideration of the urine sampling time recommended by the American Conference Governmental Industrial Hygienists (ACGIH).

GPNA inhibits the sodium-independent transport system L for neutral amino acids.

L-γ-Glutamyl-p-nitroanilide (GPNA) is widely used to inhibit the glutamine transporter ASCT2, although it is known that it also inhibits other sodium-dependent amino acid transporters. In a panel of human cancer cell lines, which express the system L transporters LAT1 and LAT2, GPNA inhibits the sodium-independent influx of leucine and glutamine. The kinetics of the effect suggests that GPNA is a low affinity, competitive inhibitor of system L transporters. In Hs683 human oligodendroglioma cells, the incubation in the presence of GPNA, but not ASCT2 silencing, lowers the cell content of leucine. Under the same conditions the activity of mTORC1 is inhibited. Decreased cell content of branched chain amino acids and mTORC1 inhibition are observed in most of the other cell lines upon incubation with GPNA. It is concluded that GPNA hinders the uptake of essential amino acids through system L transporters and lowers their cell content.

Dependence on glutamine uptake and glutamine addiction characterize myeloma cells: a new attractive target.

The importance of glutamine (Gln) metabolism in multiple myeloma (MM) cells and its potential role as a therapeutic target are still unknown, although it has been reported that human myeloma cell lines (HMCLs) are highly sensitive to Gln depletion. In this study, we found that both HMCLs and primary bone marrow (BM) CD138(+) cells produced large amounts of ammonium in the presence of Gln. MM patients have lower BM plasma Gln with higher ammonium and glutamate than patients with indolent monoclonal gammopathies. Interestingly, HMCLs expressed glutaminase (GLS1) and were sensitive to its inhibition, whereas they exhibited negligible expression of glutamine synthetase (GS). High GLS1 and low GS expression were also observed in primary CD138(+) cells. Gln-free incubation or treatment with the glutaminolytic enzyme l-asparaginase depleted the cell contents of Gln, glutamate, and the anaplerotic substrate 2-oxoglutarate, inhibiting MM cell growth. Consistent with the dependence of MM cells on extracellular Gln, a gene expression profile analysis, on both proprietary and published datasets, showed an increased expression of the Gln transporters SNAT1, ASCT2, and LAT1 by CD138(+) cells across the progression of monoclonal gammopathies. Among these transporters, only ASCT2 inhibition in HMCLs caused a marked decrease in Gln uptake and a significant fall in cell growth. Consistently, stable ASCT2 downregulation by a lentiviral approach inhibited HMCL growth in vitro and in a murine model. In conclusion, MM cells strictly depend on extracellular Gln and show features of Gln addiction. Therefore, the inhibition of Gln uptake is a new attractive therapeutic strategy for MM.

Effect of ABCG2/BCRP Expression on Efflux and Uptake of Gefitinib in NSCLC Cell Lines.

BACKGROUND: BCRP/ABCG2 emerged as an important multidrug resistance protein, because it confers resistance to several classes of cancer chemotherapeutic agents and to a number of novel molecularly-targeted therapeutics such as tyrosine kinase inhibitors. Gefitinib is an orally active, selective EGFR tyrosine kinase inhibitor used in the treatment of patients with advanced non small cell lung cancer (NSCLC) carrying activating EGFR mutations. Membrane transporters may affect the distribution and accumulation of gefitinib in tumour cells; in particular a reduced intracellular level of the drug may result from poor uptake, enhanced efflux or increased metabolism. AIM: The present study, performed in a panel of NSCLC cell lines expressing different ABCG2 plasma membrane levels, was designed to investigate the effect of the efflux transporter ABCG2 on intracellular gefitinib accumulation, by dissecting the contribution of uptake and efflux processes. METHODS AND RESULTS: Our findings indicate that gefitinib, in lung cancer cells, inhibits ABCG2 activity, as previously reported. In addition, we suggest that ABCG2 silencing or overexpression affects intracellular gefitinib content by modulating the uptake rather than the efflux. Similarly, overexpression of ABCG2 affected the expression of a number of drug transporters, altering the functional activities of nutrient and drug transport systems, in particular inhibiting MPP, glucose and glutamine uptake. CONCLUSIONS: Therefore, we conclude that gefitinib is an inhibitor but not a substrate for ABCG2 and that ABCG2 overexpression may modulate the expression and activity of other transporters involved in the uptake of different substrates into the cells.

Quantification of 3-MCPD and its mercapturic metabolite in human urine: validation of an LC-MS-MS method and its application in the general population.

A new method for the simultaneous quantitative determination in human urine of 3-monochloropropane-1,2-diol (3-MCPD), a toxic food contaminant, and its metabolite, 2,3-dihydroxypropyl mercapturic acid (DHPMA), was developed and validated. After urine dilution, the analytes were separated on an Atlantis®dC18 column and quantified by liquid chromatography-tandem mass spectrometry using isotopically labelled internal standards. The limits of quantification (S/N = 10) were 1.90 and 2.21 µg/L for 3-MCPD and DHPMA, respectively. Intra- and inter-day precision were lower than 6 % for each compound. Matrix effects were evaluated. Due to the high sensitivity and good accuracy of the method, 3-MCPD and DHPMA were found in 67 and 100 % of urine samples of healthy subjects, respectively.

N-3 PUFA increase bioavailability and function of endothelial progenitor cells.

BACKGROUND AND AIMS: Recent data suggest that n-3 PUFA exert beneficial effects on endothelial progenitor cell (EPC) biology. We sought to investigate whether these effects might be mediated by enhanced EPC in vitro function and/or in vivo bioavailability. METHODS AND RESULTS: CACs and late-outgrowth EPCs were isolated from peripheral blood mononuclear cells obtained from 12 donor buffy-coats. The effect of n-3 PUFA (EPA:DHA = 0.9:1.5; 9 µM EPA plus 15 µM DHA) was tested on CAC/EPC viability, function (tube-formation) and pro-inflammatory molecule expression. Circulating EPC (cells positive for CD34, CD133 and kinase insert domain receptor - KDR cell-surface antigens by flow cytometry) number was evaluated in 20 healthy subjects (10 F/10 M, 32 ± 5 years), randomized to receive 4 mackerel or sardine portions per week for 6 weeks followed by a 6 week free-diet period. N-3 PUFA improved CAC and late-outgrowth EPC viability (p < 0.05) and the capacity to form tube-like structures in CACs (+38%; p < 0.05) and late-outgrowth EPCs (+15%; p < 0.05). ICAM-1 expression was reduced in both CACs (p < 0.05) and late-outgrowth EPCs (p < 0.05) and VCAM-1 in late-outgrowth EPCs (p < 0.005). N-3 PUFA significantly decreased TNF-α and MCP-1 expression in CACs and IL-8, TNF-α and MCP-1 in late-outgrowth EPCs (p < 0.05). Circulating EPC number significantly improved after 6 weeks of a fish-enriched diet (p < 0.01) and returned to baseline levels after a 6 week free-diet period (p < 0.01). Plasma EPA levels were independently and positively associated with EPC levels (p < 0.005). CONCLUSION: Our findings support the case of a beneficiary role played by n-3 PUFA in EPC function and bioavailability.

Persistent lone atrial fibrillation: clinicopathologic study of 19 cases.

BACKGROUND: The extent to which atrial myocardium is remodeled in patients with persistent lone atrial fibrillation (LAF) is largely unknown. OBJECTIVE: The purpose of this study was to perform a clinicopathologic investigation in patients with persistent LAF. METHODS: We characterized structural and molecular remodeling in atrial biopsies from 19 patients (17 males, mean age 49 years) with persistent (>7 days; n = 8) or long-lasting persistent (>1 year; n = 11) LAF who underwent surgical ablation. Atrial tissue from 15 autopsy samples without clinicopathologic evidence of heart disease served as controls. RESULTS: Morphometric analysis showed cardiomyocyte hypertrophy and greater amounts of myolytic damage and interstitial fibrosis in persistent LAF patients compared to controls (P <.0001). Atrial tissue levels of heme oxygenase-1 and 3-nitrotyrosine were increased in persistent LAF patients (P <.001), consistent with oxidative stress. Levels of superoxide dismutase-2, interleukin-8, interleukin-10, tumor necrosis factor-α, and thiobarbituric acid reactive substance were greater in controls than in persistent LAF patients. Immunoreactive signal for connexin43 was reduced more frequently in persistent LAF patients than controls. There was no correlation between features of structural or molecular remodeling and clinical parameters, including persistent LAF duration. CONCLUSION: In persistent LAF patients, the atria are modified by structural remodeling and molecular changes of oxidative stress. Tissue changes in persistent LAF appear to occur early after its onset and are qualitatively no different than those observed in patients with atrial fibrillation related to conventional risk factors. These findings suggest that different types of atrial fibrillation are associated with the same spectrum of tissue lesions. Early intervention to restore sinus rhythm in persistent LAF patients may prevent irreversible tissue change, especially interstitial fibrosis.

Correlation between erlotinib pharmacokinetics, cutaneous toxicity and clinical outcomes in patients with advanced non-small cell lung cancer (NSCLC).

OBJECTIVES: An association between skin toxicity and outcome has been reported for NSCLC patients treated with erlotinib. Several explanations have been suggested, including pharmacokinetic and pharmacogenomic variability. The purposes of this study were to characterize erlotinib pharmacokinetic and to correlate drug serum and urine levels to toxicity and outcomes in advanced NSCLC patients. METHODS: Patients with stage IV NSCLC consecutively treated with erlotinib in second- or third-line were enrolled. Biological samples (blood, urine and tumor specimens) were collected. Erlotinib levels in serum and urine samples of all patients after 7 (T1) and 30 (T2) days of treatment were quantified by LC-MS/MS analysis, along with urinary 6ß-hydroxycortisol/cortisol ratio, as marker of metabolic phenotype of the CYP3A4/5 enzyme. RESULTS: 56 patients were recruited and for 46 all samples were available. At T1 erlotinib levels were 3.90 [2.13] µmol/l and 0.37 [2.90]µmol/mol creat in serum and urinary samples, respectively; at T2 drug concentrations were significantly lower (2.02 [4.05] µmol/l and 0.23 [4.47] µmol/mol creat, respectively). Patients with grade 3 skin toxicity showed serum T1 drug levels significantly higher than those with grade 0-2 (6.84 [2.28] vs. 3.08 [1.97] µmol/l, respectively, p=0.004) and had longer progression-free and overall survival. An inverse correlation between erlotinib serum levels and urinary 6ß-hydroxycortisol/cortisol ratio was observed in patients with grade 3 skin toxicity. CONCLUSIONS: These findings suggest that the pharmacokinetics and metabolism of erlotinib are related to skin toxicity and may support further studies where erlotinib dosing is tailored according to pharmacokinetic parameters.

[Monitoring of the occupational and environmental exposure to low doses of benzene]. / Il monitoraggio dell'esposizione occupazionale ed ambientale a basse dosi di benzene.

AIM: To verify which of the various biomarkers of internal dose of benzene can be considered reliable for biological monitoring of exposure to the low concentrations present nowadays in working and living environments. MATERIALS AND METHODS: The specific literature was analyzed to assess the reliability of the different biomarkers of internal dose. RESULTS AND CONCLUSIONS: T,t-muconic acid is a non specific biomarker for benzene, valid for exposure to concentrations up to one order of magnitude less than the threshold limit of 3250 microg/m3. S-phenylmercapturic acid (SPMA) is a reliable marker even for exposure to concentrations up to two orders below the threshold value of 3250 microg/m3, and can be considered the biomarker of choice for biological monitoring of workers exposed to benzene. Urinary benzene does not seem to have any real advantages over SPMA for monitoring occupational exposure to benzene, but it does seem to be more reliable than SPMA to assess exposure to concentrations like those present in living environments. A smoking habit influences the urinary excretion of all the described biomarkers, and for the current low levels of occupational and environmental exposure to benzene, must be taken into account when interpreting the results of biological monitoring.