Biological monitoring of chemical exposure : toxicokinetic differences due to age and sex

Human biomonitoring is a widely used method in the assessment of occupational exposure to chemical substances and recommended biological limits are published periodically for interpretation and decision-making. However, it is increasingly recognized that a large variability is associated with biological monitoring, making interpretation less efficient than assumed. In order to improve the applicability of biological monitoring, specific factors responsible for this variability should be identified and their contribution quantified. Among these factors, age and sex are easily identifiable, and present knowledge about pharmaceutical chemicals suggests that they play an important role on the toxicokinetics of occupational chemical agents, and therefore on the biological monitoring results.The aim of the present research project was to assess the influence of age and sex on biological indicators corresponding to organic solvents. This has been done experimentally and by toxicokinetic computer simulation. Another purpose was to explore the effect of selected CYP2E1 polymorphisms on the toxicokinetic profile.Age differences were identified by numerical simulations using a general toxicokinetic model from a previous study which was applied to 14 chemicals, representing 21 specific biological entities, with, among others, toluene, phenol, lead and mercury. These models were runn with the modified parameters, indicating in some cases important differences due to age. The expected changes are mostly of the order of 10-20 %, but differences up to 50 % were observed in some cases. These differences appear to depend on the chemical and on the biological entity considered.Sex differences were quantified by controlled human exposures, which were carried out in a 12 m3 exposure chamber for three organic solvents separately: methyl ethyl ketone, 1-methoxy-2-propanol and 1,1,1-trichloroethane. The human volunteer groups were composed 12 of ten young men and fifteen young women, the latter subdivided into those with and without hormonal contraceptive. They were exposed during six hours at rest and at half of the threshold limit value. The kinetics of the parent compounds (organic volatiles) and their metabolite(s) were followed in blood, urine and expired air over time. Analyses of the solvent and their metabolites were performed by using headspace gas chromatography, CYP2E1 genotypes by using PCR-based RFLP methods. Experimental data were used to calibrate the toxicokinetic models developed for the three solvents. The results obtained for the different biomarkers of exposure mainly showed an effect on the urinary levels of several biomarkers among women due to the use of hormonal contraceptive, with an increase of about 50 % in the metabolism rate. The results also showed a difference due to the genotype CYP2E1*6, when exposed to methyl ethyl ketone, with a tendency to increase CYP2E1 activity when volunteers were carriers of the mutant allele. Simulations showed that it is possible to use simple toxicokinetic tools in order to predict internal exposure when exposed to organic solvents. Our study suggests that not only physiological differences but also exogenous sex hormones could influence CYP2E1 enzyme activity. The variability among the urinary biological indicators levels gives evidence of an interindividual susceptibility, an aspect that should have its place in the approaches for setting limits of occupational exposure.

LB11058, a new cephalosporin with high penicillin-binding protein 2a affinity and activity in experimental endocarditis due to homogeneously methicillin-resistant Staphylococcus aureus.

LB11058 is a new synthetic cephalosporin with good affinity for staphylococcal penicillin-binding protein 2a (PBP2a). LB11058 was tested in vitro and in rats with experimental aortic endocarditis against three methicillin-resistant Staphylococcus aureus (MRSA) strains, one penicillinase-negative strain (strain COL), and two penicillinase-producing strains (COL-Bla+ and P8-Hom). The MICs of LB11058 for the organisms were 1 mg/liter. The MICs of vancomycin and ceftriaxone were 1 and >/=64 mg/liter, respectively. In population analysis profiles, none of the MRSA strains grew at >/=2 mg of LB11058/liter. Rats with endocarditis were treated for 5 days. LB11058 was highly bound to serum proteins in rats (>/=98%). However, binding was saturable above a threshold of 250 mg/liter. Therefore, continuous concentrations of 250 mg/liter in serum were infused to ensure a free fraction (>/=5 mg/liter) above the drug's MIC for the entire infusion period. Control treatments included simulation of human serum kinetics produced by intravenous vancomycin (1 g twice daily, free drug concentration above MIC, >/=90% of infusion period) or ceftriaxone (2 g/24 h, free drug concentrations above the MIC, 0% of infusion period). LB11058 successfully treated 10 of 10 (100%) and 13 of 14 (93%) of rats infected with COL-Bla+ and P8-Hom, respectively. This was comparable to vancomycin (sterilization of 8 of 12 [66%] and 6 of 8 [75%] rats, respectively). Ceftriaxone was inactive. Low concentrations of LB11058 (5 and 10 mg/liter, continuously infused) in serum were ineffective, as predicted by the pharmacodynamic parameters. At appropriate doses, LB11058 was highly effective both in vitro and in vivo. This finding supports the development of this beta-lactam with high PBP2a affinity for the treatment of MRSA infections.

Effects of parental larval diet on egg size and offspring traits in Drosophila.

If a mother's nutritional status predicts the nutritional environment of the offspring, it would be adaptive for mothers experiencing nutritional stress to prime their offspring for a better tolerance to poor nutrition. We report that in Drosophila melanogaster, parents raised on poor larval food laid 3-6% heavier eggs than parents raised on standard food, despite being 30 per cent smaller. Their offspring developed 14 h (4%) faster on the poor food than offspring of well-fed parents. However, they were slightly smaller as adults. Thus, the effects of parental diet on offspring performance under malnutrition apparently involve both adaptive plasticity and maladaptive effects of parental stress.

Parenteral anticoagulants in heart disease: Current status and perspectives (Section II). Position Paper of the ESC Working Group on Thrombosis - Task Force on Anticoagulants in Heart Disease.

Anticoagulants are a mainstay of cardiovascular therapy, and parenteral anticoagulants have widespread use in cardiology, especially in acute situations. Parenteral anticoagulants include unfractionated heparin, low-molecular-weight heparins, the synthetic pentasaccharides fondaparinux, idraparinux and idrabiotaparinux, and parenteral direct thrombin inhibitors. The several shortcomings of unfractionated heparin and of low-molecular-weight heparins have prompted the development of the other newer agents. Here we review the mechanisms of action, pharmacological properties and side effects of parenteral anticoagulants used in the management of coronary heart disease treated with or without percutaneous coronary interventions, cardioversion for atrial fibrillation, and prosthetic heart valves and valve repair. Using an evidence-based approach, we describe the results of completed clinical trials, highlight ongoing research with currently available agents, and recommend therapeutic options for specific heart diseases.

Fitness cost and impaired survival in penicillin-resistant Streptococcus gordonii isolates selected in the laboratory.

Cycling of Streptococcus gordonii (115 times) with penicillin resulted in a MIC increase of more than 100-fold, from 0.008 to 2 microg/ml. The 2-microg/ml MIC maximum was already reached after 36 passages but resulted in impaired fitness. Although the MIC did not increase further, fitness was partially recovered during the 79 additional cycles.

Activities of fosfomycin and rifampin on planktonic and adherent Enterococcus faecalis strains in an experimental foreign-body infection model.

Enterococcal implant-associated infections are difficult to treat because antibiotics generally lack activity against enterococcal biofilms. We investigated fosfomycin, rifampin, and their combinations against planktonic and adherent Enterococcus faecalis (ATCC 19433) in vitro and in a foreign-body infection model. The MIC/MBClog values were 32/>512 μg/ml for fosfomycin, 4/>64 μg/ml for rifampin, 1/2 μg/ml for ampicillin, 2/>256 μg/ml for linezolid, 16/32 μg/ml for gentamicin, 1/>64 μg/ml for vancomycin, and 1/5 μg/ml for daptomycin. In time-kill studies, fosfomycin was bactericidal at 8× and 16× MIC, but regrowth of resistant strains occurred after 24 h. With the exception of gentamicin, no complete inhibition of growth-related heat production was observed with other antimicrobials on early (3 h) or mature (24 h) biofilms. In the animal model, fosfomycin alone or in combination with daptomycin reduced planktonic counts by ≈4 log10 CFU/ml below the levels before treatment. Fosfomycin cleared planktonic bacteria from 74% of cage fluids (i.e., no growth in aspirated fluid) and eradicated biofilm bacteria from 43% of cages (i.e., no growth from removed cages). In combination with gentamicin, fosfomycin cleared 77% and cured 58% of cages; in combination with vancomycin, fosfomycin cleared 33% and cured 18% of cages; in combination with daptomycin, fosfomycin cleared 75% and cured 17% of cages. Rifampin showed no activity on planktonic or adherent E. faecalis, whereas in combination with daptomycin it cured 17% and with fosfomycin it cured 25% of cages. Emergence of fosfomycin resistance was not observed in vivo. In conclusion, fosfomycin showed activity against planktonic and adherent E. faecalis. Its role against enterococcal biofilms should be further investigated, especially in combination with rifampin and/or daptomycin treatment.

New drugs and combinations for malignant glioma.

A new chemotherapy agent and a method for local delivery of carmustine have recently been approved for the treatment of malignant glioma. However, the increase in survival remains modest at best with only a very select patients currently benefiting truly of these treatments. Combination regimen of different alkylating agents or prior O6-alkyltransferase depletion by O6-benzylguanine or continuous temozolomide administration schedules have shown some indication for increased activity. There is preclinical rational for combining temozolomide with radiotherapy and the initial results of a phase II clinical trial were promising. Several new cytotoxic agents are currently in clinical trials in patients with recurrent glioma. More importantly, targeted therapy and antiangiogenic agents have entered the clinical development phase also for patients with glioblastoma and anaplastic astrocytoma. The optimal timing of administration of non-cytotoxic substances and their integration into the currently available treatments remains a challenge. Novel study designs and identification of surrogate markers are necessary in order to make rapid and clinically meaningful progress. This review summarises the currently available evidence of activity of the recently approved drugs against malignant glioma and mentions also agents which have failed to demonstrate a significant antitumour activity. Study endpoints are critically discussed. Combination regimens with other agents and radiation therapy are reviewed. The rational for using antiangiogenic drugs in selected ongoing trials is discussed.

Ultra-performance liquid chromatography mass spectrometry and sensitive bioassay methods for quantification of posaconazole plasma concentrations after oral dosing.

Posaconazole (POS) is a new antifungal agent for prevention and therapy of mycoses in immunocompromised patients. Variable POS pharmacokinetics after oral dosing may influence efficacy: a trough threshold of 0.5 ?g/ml has been recently proposed. Measurement of POS plasma concentrations by complex chromatographic techniques may thus contribute to optimize prevention and management of life-threatening infections. No microbiological analytical method is available. The objective of this study was to develop and validate a new simplified ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method and a sensitive bioassay for quantification of POS over the clinical plasma concentration range. The UPLC-MS/MS equipment consisted of a triple quadrupole mass spectrometer, an electrospray ionization (ESI) source, and a C(18) analytical column. The Candida albicans POS-hypersusceptible mutant (MIC of 0.002 ?g/ml) ?cdr1 ?cdr2 ?flu ?mdr1 ?can constructed by targeted deletion of multidrug efflux transporters and calcineurin genes was used for the bioassay. POS was extracted from plasma by protein precipitation with acetonitrile-methanol (75%/25%, vol/vol). Reproducible standard curves were obtained over the range 0.014 to 12 (UPLC-MS/MS) and 0.028 to 12 ?g/ml (bioassay). Intra- and interrun accuracy levels were 106% ± 2% and 103% ± 4% for UPLC-MS/MS and 102% ± 8% and 104% ± 1% for bioassay, respectively. The intra- and interrun coefficients of variation were 7% ± 4% and 7% ± 3% for UPLC-MS/MS and 5% ± 3% and 4% ± 2% for bioassay, respectively. An excellent correlation between POS plasma concentrations measured by UPLC-MS/MS and bioassay was found (concordance, 0.96). In 26 hemato-oncological patients receiving oral POS, 27/69 (39%) trough plasma concentrations were lower than 0.5 ?g/ml. The UPLC-MS/MS method and sensitive bioassay offer alternative tools for accurate and precise quantification of the plasma concentrations in patients receiving oral posaconazole.

In vitro characterization of PlySK1249, a novel phage lysin, and assessment of its antibacterial activity in a mouse model of Streptococcus agalactiae bacteremia.

Beta-hemolytic Streptococcus agalactiae is the leading cause of bacteremia and invasive infections. These diseases are treated with β-lactams or macrolides, but the emergence of less susceptible and even fully resistant strains is a cause for concern. New bacteriophage lysins could be promising alternatives against such organisms. They hydrolyze the bacterial peptidoglycan at the end of the phage cycle, in order to release the phage progeny. By using a bioinformatic approach to screen several beta-hemolytic streptococci, a gene coding for a lysin was identified on a prophage carried by Streptococcus dysgalactiae subsp. equisimilis SK1249. The gene product, named PlySK1249, harbored an original three-domain structure with a central cell wall-binding domain surrounded by an N-terminal amidase and a C-terminal CHAP domain. Purified PlySK1249 was highly lytic and bactericidal for S. dysgalactiae (2-log10 CFU/ml decrease within 15 min). Moreover, it also efficiently killed S. agalactiae (1.5-log10 CFU/ml decrease within 15 min) but not several streptococcal commensal species. We further investigated the activity of PlySK1249 in a mouse model of S. agalactiae bacteremia. Eighty percent of the animals (n = 10) challenged intraperitoneally with 10(6) CFU of S. agalactiae died within 72 h, whereas repeated injections of PlySK1249 (45 mg/kg 3 times within 24 h) significantly protected the mice (P < 0.01). Thus, PlySK1249, which was isolated from S. dysgalactiae, demonstrated high cross-lytic activity against S. agalactiae both in vitro and in vivo. These encouraging results indicated that PlySK1249 might represent a good candidate to be developed as a new enzybiotic for the treatment of systemic S. agalactiae infections.

The potential impact of new effervescent alendronate formulation on compliance and persistence in osteoporosis treatment.

Osteoporotic fractures are a public health problem and their incidence and subsequent economic and social costs are expected to rise in the next future. Different drugs have been developed to reduce osteoporosis and the risk of osteoporotic fractures, and among them, antiresorptive agents, and in particular oral alendronate, are the most widely utilized. However, one of the most common problems with antiresorptive drugs is poor adherence to treatment, which is associated with a high fracture incidence and with an increase in hospitalization costs. One of the main reasons of poor adherence to these treatments is the occurrence of adverse events, mainly at gastrointestinal (GI) level, including dyspepsia, dysphagia, and esophageal ulcers. In light of these considerations the aim of this paper is to perform a literature review to show the pathophysiologic bases of GI alendronate-induced adverse events and how new bisphosphonate formulations like effervescent alendronate can improve compliance and persistence to treatment and decrease the fracture rate incidence in osteoporotic patients.

Functional characterization of the Pneumocystis jirovecii potential drug targets dhfs and abz2 involved in folate biosynthesis.

Pneumocystis species are fungal parasites colonizing mammal lungs with strict host specificity. Pneumocystis jirovecii is the human-specific species and can turn into an opportunistic pathogen causing severe pneumonia in immunocompromised individuals. This disease is currently the second most frequent life-threatening invasive fungal infection worldwide. The most efficient drug, cotrimoxazole, presents serious side effects, and resistance to this drug is emerging. The search for new targets for the development of new drugs is thus of utmost importance. The recent release of the P. jirovecii genome sequence opens a new era for this task. It can now be carried out on the actual targets to be inhibited instead of on those of the relatively distant model Pneumocystis carinii, the species infecting rats. We focused on the folic acid biosynthesis pathway because (i) it is widely used for efficient therapeutic intervention, and (ii) it involves several enzymes that are essential for the pathogen and have no human counterparts. In this study, we report the identification of two such potential targets within the genome of P. jirovecii, the dihydrofolate synthase (dhfs) and the aminodeoxychorismate lyase (abz2). The function of these enzymes was demonstrated by the rescue of the null allele of the orthologous gene of Saccharomyces cerevisiae.

Present standards and future perspectives in the treatment of metastatic non-small cell lung cancer.

The development of novel effective immunotherapeutic agents and early clinical data hinting at significant activity in non-small cell lung cancer (NSCLC) has introduced yet another player in the field of management of advanced disease. At present, first-line cytotoxic chemotherapy is generally withheld pending results of molecular testing for any actionable genetic alteration that could lead to targeted treatment, and in their absence chemotherapy is prescribed as a default therapy. Phase III trials comparing head-to-head immune checkpoint inhibitors with standard platinum-based doublet chemotherapy are underway. Second-line chemotherapy is likewise being challenged in phase III trials, one of which having recently reported positive results in advanced squamous cell carcinoma. In tumors harboring actionable transforming genetic alterations such as EGFR mutations and ALK rearrangements, second- and third-generation inhibitors allow for multiple lines of targeted treatment beyond initial resistance, postponing the use of cytotoxic chemotherapy to very late lines of therapy. Chemotherapy as a longstanding but still present standard of care capable of prolonging survival, improving quality of life, and relieving symptoms sees its role increasingly restricted to clinical, immunological, and molecular subsets of patients where its activity and efficacy have never been tested prospectively.

Hsp70 and the Cochaperone StiA (Hop) Orchestrate Hsp90-Mediated Caspofungin Tolerance in Aspergillus fumigatus.

Aspergillus fumigatus is the primary etiologic agent of invasive aspergillosis (IA), a major cause of death among immunosuppressed patients. Echinocandins (e.g., caspofungin) are increasingly used as second-line therapy for IA, but their activity is only fungistatic. Heat shock protein 90 (Hsp90) was previously shown to trigger tolerance to caspofungin and the paradoxical effect (i.e., decreased efficacy of caspofungin at higher concentrations). Here, we demonstrate the key role of another molecular chaperone, Hsp70, in governing the stress response to caspofungin via Hsp90 and their cochaperone Hop/Sti1 (StiA in A. fumigatus). Mutation of the StiA-interacting domain of Hsp70 (C-terminal EELD motif) impaired thermal adaptation and caspofungin tolerance with loss of the caspofungin paradoxical effect. Impaired Hsp90 function and increased susceptibility to caspofungin were also observed following pharmacologic inhibition of the C-terminal domain of Hsp70 by pifithrin-μ or after stiA deletion, further supporting the links among Hsp70, StiA, and Hsp90 in governing caspofungin tolerance. StiA was not required for the physical interaction between Hsp70 and Hsp90 but had distinct roles in the regulation of their function in caspofungin and heat stress responses. In conclusion, this study deciphering the physical and functional interactions of the Hsp70-StiA-Hsp90 complex provided new insights into the mechanisms of tolerance to caspofungin in A. fumigatus and revealed a key C-terminal motif of Hsp70, which can be targeted by specific inhibitors, such as pifithrin-μ, to enhance the antifungal activity of caspofungin against A. fumigatus.

Calcium-Mediated Induction of Paradoxical Growth following Caspofungin Treatment Is Associated with Calcineurin Activation and Phosphorylation in Aspergillus fumigatus.

The echinocandin antifungal drug caspofungin at high concentrations reverses the growth inhibition of Aspergillus fumigatus, a phenomenon known as the "paradoxical effect," which is not consistently observed with other echinocandins (micafungin and anidulafungin). Previous studies of A. fumigatus revealed the loss of the paradoxical effect following pharmacological or genetic inhibition of calcineurin, yet the underlying mechanism is poorly understood. Here, we utilized a codon-optimized bioluminescent Ca(2+) reporter aequorin expression system in A. fumigatus and showed that caspofungin elicits a transient increase in cytosolic free Ca(2+) ([Ca(2+)]c) in the fungus that acts as the initial trigger of the paradoxical effect by activating calmodulin-calcineurin signaling. While the increase in [Ca(2+)]c was also observed upon treatment with micafungin, another echinocandin without the paradoxical effect, a higher [Ca(2+)]c increase was noted with the paradoxical-growth concentration of caspofungin. Treatments with a Ca(2+)-selective chelator, BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid], or the L-type Ca(2+) channel blocker verapamil abolished caspofungin-mediated paradoxical growth in both the wild-type and the echinocandin-resistant (EMFR-S678P) strains. Concomitant with increased [Ca(2+)]c levels at higher concentrations of caspofungin, calmodulin and calcineurin gene expression was enhanced. Phosphoproteomic analysis revealed that calcineurin is activated through phosphorylation at its serine-proline-rich region (SPRR), a domain previously shown to be essential for regulation of hyphal growth, only at a paradoxical-growth concentration of caspofungin. Our results indicate that as opposed to micafungin, the increased [Ca(2+)]c at high concentrations of caspofungin activates calmodulin-calcineurin signaling at both a transcriptional and a posttranslational level and ultimately leads to paradoxical fungal growth.