Chemical warfare agent simulants for human volunteer trials of emergency decontamination: A systematic review.

Incidents involving the release of chemical agents can pose significant risks to public health. In such an event, emergency decontamination of affected casualties may need to be undertaken to reduce injury and possible loss of life. To ensure these methods are effective, human volunteer trials (HVTs) of decontamination protocols, using simulant contaminants, have been conducted. Simulants must be used to mimic the physicochemical properties of more harmful chemicals, while remaining non-toxic at the dose applied. This review focuses on studies that employed chemical warfare agent simulants in decontamination contexts, to identify those simulants most suitable for use in HVTs of emergency decontamination. Twenty-two simulants were identified, of which 17 were determined unsuitable for use in HVTs. The remaining simulants (n = 5) were further scrutinized for potential suitability according to toxicity, physicochemical properties and similarities to their equivalent toxic counterparts. Three suitable simulants, for use in HVTs were identified; methyl salicylate (simulant for sulphur mustard), diethyl malonate (simulant for soman) and malathion (simulant for VX or toxic industrial chemicals). All have been safely used in previous HVTs, and have a range of physicochemical properties that would allow useful inference to more toxic chemicals when employed in future studies of emergency decontamination systems.

Involvement of phosphoinositide 3-kinase and Akt in the induction of muscle protein degradation by proteolysis-inducing factor.

In the present study the role of Akt/PKB (protein kinase B) in PIF- (proteolysis-inducing factor) induced protein degradation has been investigated in murine myotubes. PIF induced transient phosphorylation of Akt at Ser(473) within 30 min, which was attenuated by the PI3K (phosphoinositide 3-kinase) inhibitor LY294002 and the tyrosine kinase inhibitor genistein. Protein degradation was attenuated in myotubes expressing a dominant-negative mutant of Akt (termed DNAkt), compared with the wild-type variant, whereas it was enhanced in myotubes containing a constitutively active Akt construct (termed MyrAkt). A similar effect was observed on the induction of the ubiquitin-proteasome pathway. Phosphorylation of Akt has been linked to up-regulation of the ubiquitin-proteasome pathway through activation of NF-kappaB (nuclear factor kappaB) in a PI3K-dependent process. Protein degradation was attenuated by rapamycin, a specific inhibitor of mTOR (mammalian target of rapamycin), when added before, or up to 30 min after, addition of PIF. PIF induced transient phosphorylation of mTOR and the 70 kDa ribosomal protein S6 kinase. These results suggest that transient activation of Akt results in an increased protein degradation through activation of NF-kappaB and that this also allows for a specific synthesis of proteasome subunits.

Identification and characterization of a membrane receptor for proteolysis-inducing factor on skeletal muscle.

Proteolysis-inducing factor (PIF) is a sulfated glycoprotein produced by cachexia-inducing tumors, which induces atrophy of skeletal muscle. PIF has been shown to bind specifically with high affinity (K(d), in nanomolar) to sarcolemma membranes from skeletal muscle of both the mouse and the pig, as well as murine myoblasts and a human muscle cell line. Ligand binding was abolished after enzymatic deglycosylation, suggesting that binding was mediated through the oligosaccharide chains in PIF. Chondroitin sulfate, but not heparan or dermatan sulfate, showed competitive inhibition (K(d), 1.1 x 10(-7) mol/L) of binding of PIF to the receptor, suggesting an interaction with the sulfated oligosaccharide chains. Ligand blotting of [(35)S]PIF to triton solublized membranes from C(2)C(12) cells provided evidence for a binding protein of apparent M(r) of approximately 40,000. Amino acid sequence analysis showed the PIF receptor to be a DING protein. Antisera reactive to a 19mer from the N-terminal amino acid residues of the binding protein attenuated protein degradation and activation of the ubiquitin-proteasome pathway induced by PIF in murine myotubes. In addition, the antisera was highly effective in attenuating the decrease in body weight in mice bearing the MAC16 tumor, with a significant increase in muscle wet weight due to an increase in the rate of protein synthesis, together with a reduction in protein degradation through attenuation of the increased proteasome expression and activity. These results confirm that the PIF binding protein has a functional role in muscle protein atrophy in cachexia and that it represents a potential new therapeutic target.

Mechanism of induction of muscle protein degradation by angiotensin II.

Angiotensin I and II have been shown to directly induce protein degradation in skeletal muscle through an increased activity and expression of the ubiquitin-proteasome proteolytic pathway. This investigation determines the role of the nuclear transcription factor nuclear factor-kappaB (NF-kappaB) in this process. Using murine myotubes as a surrogate model system both angiotensin I and II were found to induce activation of protein kinase C (PKC), with a parabolic dose-response curve similar to the induction of total protein degradation. Activation of PKC was required for the induction of proteasome expression, since calphostin C, a highly specific inhibitor of PKC, attenuated both the increase in total protein degradation and in proteasome expression and functional activity increased by angiotensin II. PKC is known to activate I-kappaB kinase (IKK), which is responsible for the phosphorylation and subsequent degradation of I-kappaB. Both angiotensin I and II induced an early decrease in cytoplasmic I-kappaB levels followed by nuclear accumulation of NF-kappaB. Using an NF-kappaB luciferase construct this was shown to increase transcriptional activation of NF-kappaB regulated genes. Maximal luciferase expression was seen at the same concentrations of angiotensin I/II as those inducing protein degradation. Total protein degradation induced by both angiotensin I and II was attenuated by resveratrol, which prevented nuclear accumulation of NF-kappaB, confirming that activation of NF-kappaB was responsible for the increased protein degradation. These results suggest that induction of proteasome expression by angiotensin I/II involves a signalling pathway involving PKC and NF-kappaB.

Effect of cancer cachexia on the activity of tripeptidyl-peptidase II in skeletal muscle.

The ubiquitin-proteasome proteolytic pathway plays a major role in degradation of myofibrillar proteins in skeletal muscle during cancer cachexia. The end-product of this pathway is oligopeptides and these are degraded by the extralysomal peptidase tripeptidyl-peptidase II (TPPII) together with various aminopeptidases to form tripeptides and amino acids. To investigate if a relationship exists between the activity of the proteasome and TPPII, functional activities have been measured in gastrocnemius muscle of mice bearing the MAC16 tumour, and with varying extents of weight loss. TPPII activity was quantitated using the specific substrate Ala-Ala-Phe-7-amido-4-methylcoumarin, while proteasome activity was determined as the 'chymotrypsin-like' enzyme activity. Both proteasome proteolytic activity and TPPII activity increased in parallel with increasing weight loss, reaching a maximum at 16% weight loss, after which there was a progressive decrease in activity for both proteases with increasing weight loss. In murine myotubes, proteolysis-inducing factor, which is a sulphated glycoprotein produced by cachexia-inducing tumours, induced an increase in activity of both proteasome and TPPII, with an identical dose-response curve, and both activities were inhibited by eicosapentaenoic acid. These results suggest that the activities of both the proteasome and TPPII are regulated in a parallel manner in cancer cachexia, and that both are induced by the same factor and probably have the same intracellular signalling pathways and transcription factors.

Signalling pathways in the induction of proteasome expression by proteolysis-inducing factor in murine myotubes.

The mechanism by which the tumour product proteolysis-inducing factor (PIF) induced increased expression of the ubiquitin-proteasome proteolytic pathway was studied in C2C12 murine myotubes. PIF directly increased total protein breakdown at concentrations between 4 and 16 nM, and the effect was attenuated by eicosapentaenoic acid (EPA) and the 12/15-lipoxygenase inhibitor 2,3,5-trimethyl-6-(3-pyridylmethyl)1,4-benzoquinone (CV-6504). PIF induced an increased expression of mRNA for proteasome alpha (C2) and beta (C5) subunits over the same concentration range as that inducing protein degradation and with a maximal effect 4 h after PIF addition. The effect was attenuated by both EPA and CV-6504, suggesting the role of a lipoxygenase metabolite in the increased gene transcription. 15(S)-Hydroxyeicosatetraenoic acid [15(S)-HETE], an intermediate in intracellular signalling by PIF was shown to activate protein kinase Calpha(PKC) over the same concentration range as that inducing proteasome expression and both effects were attenuated by calphostin C, a highly specific inhibitor of PKC. 15(S)-HETE induced phosphorylation and degradation of IkappaBalpha at the same concentrations as those inducing 20S proteasome expression, and this effect was attenuated by calphostin C, suggesting the mediation of PKC. These results suggest potential control points in proteasome activation that could be useful for therapeutic intervention.

Mechanism of the attenuation of proteolysis-inducing factor stimulated protein degradation in muscle by beta-hydroxy-beta-methylbutyrate.

The leucine metabolite beta-hydroxy-beta-methylbutyrate (HMB) prevents muscle protein degradation in cancer-induced weight loss through attenuation of the ubiquitin-proteasome proteolytic pathway. To investigate the mechanism of this effect, the action of HMB on protein breakdown and intracellular signaling leading to increased proteasome expression by the tumor factor proteolysis-inducing factor (PIF) has been studied in vitro using murine myotubes as a surrogate model of skeletal muscle. A comparison has been made of the effects of HMB and those of eicosapentaenoic acid (EPA), a known inhibitor of PIF signaling. At a concentration of 50 mumol/L, EPA and HMB completely attenuated PIF-induced protein degradation and induction of the ubiquitin-proteasome proteolytic pathway, as determined by the "chymotrypsin-like" enzyme activity, as well as protein expression of 20S proteasome alpha- and beta-subunits and subunit p42 of the 19S regulator. The primary event in PIF-induced protein degradation is thought to be release of arachidonic acid from membrane phospholipids, and this process was attenuated by EPA, but not HMB, suggesting that HMB might act at another step in the PIF signaling pathway. EPA and HMB at a concentration of 50 mumol/L attenuated PIF-induced activation of protein kinase C and the subsequent degradation of inhibitor kappaBalpha and nuclear accumulation of nuclear factor kappaB. EPA and HMB also attenuated phosphorylation of p42/44 mitogen-activated protein kinase by PIF, thought to be important in PIF-induced proteasome expression. These results suggest that HMB attenuates PIF-induced activation and increased gene expression of the ubiquitin-proteasome proteolytic pathway, reducing protein degradation.