Preliminary experience with gemcitabine and cisplatin adjuvant chemotherapy after liver transplantation for hepatocellular carcinoma.

AIM: Liver transplantation (LT) criteria for treatment of hepatocellular carcinoma (HCC) were refined to improved survival and disease-free rates. Adjuvant chemotherapy might eliminate disseminated tumor cells after removal of the primary liver cancer and thereby benefit LT recipients. Our purpose was to evaluate the effect of an adjuvant chemotherapy (gemcitabine and cisplatin) on outcome of patients treated with LT for HCC. METHODS: Of the 99 patients who underwent liver transplantation from October 2001 through February 2006, there were 58 with HCC. Nine patients with extra-hepatic metastasis and four who died for noncancer-related reasons were excluded. Three groups (total n=45) were compared: Group A (n=15) met the Milan criteria and did not receive study chemotherapy, Group B (n=13) did not fit the Milan criteria and did not receive chemotherapy, and Group C (n=17) did not fit the Milan criteria and received gemcitabine and cisplatin. RESULTS: The chemotherapy regimen was well tolerated. Leukopenia, the need for granulocyte colony-stimulating factor treatment, or both occurred in four patients. The disease-specific survival rates were better for groups A and C than for group B (p=0.02) and the disease-free survival rates were also better for groups A and C than for group B (p=0.01). CONCLUSIONS: Systemic gemcitabine and cisplatin may improve disease-specific and disease-free survival in HCC patients who do not meet the Milan criteria after LT.

Radiofrequency ablation after transarterial embolization as therapy for patients with unresectable hepatocellular carcinoma.

PURPOSE: To evaluate the usefulness of transcatheter arterial embolization (TAE) followed by radiofrequency ablation (RFA) as combined treatment for unresectable hepatocellular carcinoma (HCC). PATIENTS AND METHODS: Thirty-six consecutive patients (cirrhosis, Child-Pugh class A or B) with solitary or oligonodular HCC were treated (41 lesions; mean size, 58.9 mm; range, 30-120 mm). RFA was performed after one TAE treatment. Local efficacy was evaluated with multiphasic computed tomography (CT) performed an average of two months after RFA and once during later follow-up. RESULTS: The mean follow-up period was 16 months (range, 2-45 months). Technical success (namely, complete tumor devascularization during the arterial phase) was achieved for 59% of lesions at the first CT evaluation and for 46% at the second evaluation. Among prognostic factors included in the analysis, only lesion diameter (< 50 mm versus > or = 50 mm) was statistically significant in terms of predicting local success (Fisher's exact test: 85% versus 43% at first CT, p<0.01; 70% versus 36% during follow-up, p=0.05). There were no major periprocedural complications. Kaplan-Meier analysis showed survival rates of 84% at 12 months and 57% at 24 months. CONCLUSIONS: Combined therapy--TAE then RFA--for unresectable HCC lesions in patients with cirrhosis produces a relatively high complete local response rate compared with TAE or RFA alone. Our results, considered with those from other case series, may help design prospective, randomized clinical trials to test combination therapy versus single-modality therapy in terms of risks and benefits.

Intrapericardial isolation of the inferior vena cava through a transdiaphragmatic pericardial window for tumor resection without sternotomy or thoracotomy.

AIMS: The prognosis for patients with advanced tumors invading the inferior vena cava (IVC) is dismal and surgical treatments for these tumors are challenging. A surgical approach that avoids sternotomy and thoracotomy for tumors invading the IVC even to the level of the hepatocaval junction would be extremely helpful. METHODS: The intrapericardial IVC was isolated via a transdiaphragmatic pericardial window using a transabdominal approach. Hepatectomy was then applied via an anterior approach until the IVC was seen. Total hepatic vascular exclusion was achieved by clamping the portal triad, intrapericardial IVC and infrahepatic IVC. We removed the primary tumor, the liver portion involved and the tumor thrombi, with segmental resection of the IVC. Vascular continuity was reestablished using a 20-mm-diameter polytetrafluoroethylene graft. RESULTS: Four patients with tumors invading the IVC were treated with this method. All underwent gross en-bloc tumor resections and all survived. CONCLUSION: This method for the resection of IVC tumors could avoid emboli dislodging from the tumor thrombi, prevent the complications of sternotomy, cardiopulmonary bypass and shorten operative times.

Microsomal monooxygenase activity in Tilapia (Oreochromis mossambicus) exposed to a bleached kraft mill effluent using different exposure systems.

Bleached kraft pulp and paper mill effluents (BKMEs) are known to have adverse effects on aquatic organisms. One of the effects of BKMEs is its ability to induce cytochrome P4501A activity in exposed fish. 7-Ethoxyresorufin O-deethylase (EROD) activity is the most common biomarker used to measure the mixed-function monooxygenase activity. In this study, Tilapia were exposed to BKMEs using different exposure systems and their hepatic EROD activity, as well as liver/somatic index (LSI), were determined. In the Phase I study, Tilapia treated with betaNF and a whole (100%) BKME using a static, non-renewal system exhibited statistically significant EROD induction, but LSI values were not altered. In the Phase II study, fish were either caged in the mill's fishpond with the whole effluent passing through or cultured in tanks receiving 100% of the BKME continuously using a flow-through system in the laboratory. Their EROD activities were then compared with the non-exposed fish (control). The EROD activities in both groups of fish were elevated significantly with the greatest induction being observed in the field-exposed group. The LSI values in all of the field-exposed fish were significantly greater than the control Tilapia. The EROD assay was sensitive in detecting biological changes in fish exposed to the BKME. Further studies are warranted to better understand the impacts of BKMEs on aquatic organisms in Taiwan.

Increased toxicity of textile effluents by a chlorination process using sodium hypochlorite.

Chlorinated textile effluents were tested for their toxicity using different bioassays. These assays were the Microtox assay, daphnia (Daphnia similis) 48-hr survival test, medaka embryo 14-day and juvenile 96-hr survival tests, and tilapia (Oreochromis mossambicus) juvenile 96-hr survival test. By comparing the results of toxicity tests on water samples collected at the instream prior to the chlorination process and at the outlet of the wastewater treatment facility, we found that wastewater toxicity was obviously increased by chlorination using NaOCl as the oxidant, as evidenced by the different bioassays used. Because no significant difference was observed in water chemistry, such as pH, DO, and conductivity, the induced-toxicity may be partially attributable to residue chlorine or other chlorinated compounds generated by chlorination. Future studies are warranted to identify the cause of the increase in the textile wastewater toxicity.

Analysis of the degradation compounds of chemical warfare agents using liquid chromatography/mass spectrometry.

The analysis of the degradation products of chemical warfare (CW) agents has been a challenge to analysts. The low volatility of these compounds makes them unsuitable for direct gas chromatography analysis without prior derivatization. Lack of a chromophore causes difficulties with classic detection methods after liquid chromatography separation. With the recent development of various interfaces that allow for the introduction of a liquid solvent stream into the mass spectrometer, the task of directly analyzing these compounds has become easier. For this report, we examined three different liquid chromatography/mass spectrometry (LC/MS) interfaces for their suitability for the analysis of CW degradation compounds. The interface types examined were particle beam electron impact ionization (PBI), electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). Several alkylphosphonates and thiodiglycol analogs that are produced from the degradation of organophosphorus nerve agents and sulfur mustard, respectively, were analyzed using each of the three techniques. Electron impact ionization following gas chromatography or particle beam introduction typically generates very reproducible, library-searchable mass spectra. Most of the CW breakdown compounds examined using the PBI interface did not produce a molecular ion. Despite the lack of a molecular ion, the mass spectra of the various compounds contained enough different structural information from fragment ions for the positive identification of each. The mass spectra generated using ESI are generally limited to protonated molecular ions with little or no fragmentation. For positive identification and confirmation, tandem mass spectrometry techniques quite often must be used. Many of the compounds in this study were characterized by prominent sodiated adducts along with the protonated molecular ion. Methylphosphonic acid produced protonated dimers, trimers, etc. Although the various adduct ions can be used for additional confirmation of the molecular weight of a compound, the adducts also can result in suppression of ionization of the compound and thus reduce sensitivity. Another 'soft' ionization technique that results in abundant protonated molecular ions is APCI. The mass spectra of the breakdown compounds produced using APCI were characterized generally by either a prominent protonated molecular ion or a dehydrated form of it. In addition, a number of structurally significant fragment ions were observed and their relative abundances could be adjusted by altering the APCI conditions. The data presented here indicate that each of the three techniques can be used successfully for direct liquid introduction and analysis of the non-volatile compounds produced from the degradation of CW agents. The mass spectra produced using each technique are quite different and could be utilized as additional confirmation of compound identity.

Army medical laboratory telemedicine: role of mass spectrometry in telediagnosis for chemical and biological defense.

An army medical field laboratory presently has the capability of performing standard protocols developed at the US Army Medical Research Institute of Chemical Defense for verification of nerve agent or sulfur mustard exposure. The protocols analyze hydrolysis products of chemical warfare agents using gas chromatography/mass spectrometry. Additionally, chemical warfare agents can produce alkylated or phosphorylated proteins following human exposure that have long biological half-lives and can be used as diagnostic biomarkers of chemical agent exposure. An analytical technique known as matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) currently is being examined for its potential to analyze these biomarkers. The technique is capable of detecting large biomolecules and modifications made to them. Its fast analysis time makes MALDI-TOF/MS technology suitable for screening casualties from chemical or biological attacks. Basic operation requires minimal training and the instrument has the potential to become field-portable. The limitation of the technique is that the generated data may require considerable expertise from knowledgeable personnel for consultation to ensure correct interpretation. The interaction between research scientists and field personnel in the acquisition of data and its interpretation via advanced digital telecommunication technologies can enhance rapid diagnosis and subsequently improve patient care in remote areas.

MALDI-ToF/MS as a diagnostic tool for the confirmation of sulfur mustard exposure.

The continual threat of chemical and biological warfare has prompted the need for unambiguous analytical methods for the confirmation of agent exposure. In this paper, we have investigated the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF/MS) as a diagnostic tool for this purpose. Mass spectral studies of the interaction of sulfur mustard (bis-(2-chloroethyl) sulfide, HD) with hemoglobin and metallothioneine were conducted. In vitro experiments with purified proteins were performed, using both HD and chloroethylethyl sulfide (CEES), in an effort to determine the extent of alkylation and occurrence of HD cross-linking using the MALDI-ToF/MS technique. In a typical experiment, 50 ml of 5 mM HD in acetonitrile was added to an equal volume of 0.5 mM hemoglobin in deionized water followed by vortexing and incubation at room temperature. After 24 h, the samples were analyzed by MALDI-ToF/MS. Mass spectral results indicated the presence of at least two distinct alkylation adducts for both HD and CEES experiments. These results demonstrate that MALDI-ToF/MS is a useful analytical technique to investigate the interaction of HD with biomolecules and may be employed potentially as a diagnostic tool for the confirmation of exposure to chemical warfare agents.

Development of an in vitro screening method for evaluating decontamination of sulfur mustard by reactive dermal formulations.

New barrier creams known as topical skin protectants (TSPs) recently have been formulated and demonstrated to be effective in delaying skin penetration of the blistering warfare agent sulfur mustard (HD). To further inactivate or neutralize HD, compounds that react with the toxic agent must be incorporated in the formulations, which then become reactive topical skin protectants (rTSPs). Specific and fast screening methods are necessary to assess the decontaminating activity of rTSPs. A headspace sampling technique in conjunction with thermal desorption and gas chromatography-mass spectrometry was evaluated as a potential screening method. A lower detection limit of 1 ng and a coefficient of variation of <20% were observed for the repetitive measurements of residual HD. Using this method, we evaluated a candidate rTSP. The percentage recovery of HD applied to the rTSP decreased by 35% over a 20-min time period compared with a non-rTSP. The candidate formulation showed an instant decontamination of the HD simulant dibutyl sulfide (decontamination was achieved in 2 s). The instrument set-up has the potential to accommodate multiple samples and can be automated. The method can be extended also to test reactive dermal formulations with toxic organophosphorus compounds.

Analysis and stability of the candidate sulfur mustard decontaminant S-330.

The chloroamide compound 1,3,4,6-tetrachloro-7,8-diphenyl-2, 5-diiminoglycoluril (S-330) was found to be a strong reactant in dermal formulations for the decontamination of sulfur mustard (HD). In this report, we present analytical methodologies applicable to the characterization, purity determination and quantitation of S-330 in bulk material or formulations. High-performance liquid chromatography-mass spectrometry (LC-MS) coupled with atmospheric pressure chemical ionization (APCI) interface or ultraviolet detector and nuclear magnetic spectroscopy (NMR) were used to identify and characterize S-330 and impurities in the synthetic lots or degradation products in formulations. Bulk synthesis using a chlorination process has yielded a product of 90% purity. The major impurity has been separated and identified structurally as the trichloro analog of S-330. Higher purity S-330 can be made using column chromatography, but this does not appear to be economical for large-scale production. Factors affecting the stability of S-330 in topical formulations include water content, pH, alcohols and UV light. Chloroamide S-330 decomposes at 50-60 degrees C and is not amenable for GC analysis. The HPLC technique is superior to NMR or active chlorine assay in the purity determination for S-330 in bulk material or formulations. In topical formulations containing S-330, 5-10% of water can be tolerated, but alcohols and acidic and basic conditions should be avoided.

Reactions of sulfides with S-330, a potential decontaminant of sulfur mustard in formulations.

Because the vesicant sulfur mustard (HD) remains a major chemical threat from either domestic terrorists or countries in conflict, topical preparations are being evaluated as protectants from HD exposure. The objective of this study was to evaluate the effectiveness of chloroamide S-330 as a potential reactive component in topical formulations. Therefore, the rate, mechanism and by-products of the oxidation reactions of sulfides by S-330 in solvent media or specific formulation vehicles were investigated. Using NMR, LC, LC-MS and GC-MS, the reactions of S-330 with HD, dibutyl sulfide (DBS) and methyl phenyl sulfide (MPS) were studied in acetonitrile, chloroform and perfluoropolyether (PFPE) oil. The oxidation of the three sulfides with S-330 was very rapid and completed in <4 min in acetonitrile-water or PFPE oil, but the rates of reaction in chloroform were significantly slower. In a large excess of S-330, the major products resulted from chlorination of the side chains. At a high HD/S-330 ratio, the major product was HD sulfoxide. Under both conditions, only a trace of HD sulfone, also a blistering agent, was observed. Reactions with DBS and MPS primarily gave sulfoxides and sulfones, with less side-chain chlorination. The chloroamide S-330 appeared to be a rapid and effective decontaminant of HD in either polar media or in a PFPE oil. The two alkyl and aryl sulfides are suitable simulants of HD for the initial screening and evaluation of S-330 or other similar oxidizing agents.

Multicomponent spectroscopic assay for hemoglobin and ferrihemoglobin species in methemoglobin treatment of cyanide poisoning.

Monitoring the concentrations of various hemoglobin and ferrihemoglobin species and their cyanide complexes is important in the study of the efficacy of methemoglobin-forming agents for the treatment of cyanide toxicity. In this study, the visible absorption spectra of three hemoglobin intermediates were experimentally determined and compared with computer-generated spectra. The data supported the assumption that the molar absorptivities of the intermediates are equivalent to the combined absorptivities of the component subunits. A multicomponent Fourier transform (FT)-aided full spectrum quantitation system (FSQ) to simultaneously measure hemoglobin (Hb), hemimethemoglobin (hemiMetHb), and the cyanide complex dicyanohemimethemoglobin (dicyhemiMetHb) was also evaluated. It was found that FSQ had satisfactory accuracy and precision to quantitate hemiMetHb and dicyhemiMetHb at concentrations from 2 to 20% of the total Hb, a range commonly encountered in the treatment of cyanide poisoning using methemoglobin-forming agents. The simplicity and rapid throughput of the method make it suitable for clinical evaluation studies and form the basis for the design of a portable instrument for field analysis of these species.

Analysis of hemoglobin derivatives by capillary isoelectric focusing and its application in the antidotal research of cyanide poisoning.

Cyanide toxicity can be reduced by the use of methemoglobin (MetHb) formers, and antidotal dosage is based on the extent of MetHb formation. Hemoglobin and ferrihemoglobin (MetHb, hemimethemoglobins alpha3+beta2+ and alpha2+beta3+, tetracyanmethemoglobin, and dicyanmethemoglobin) concentrations in human, pig, and mouse blood were determined after separation by isoelectric focusing with an octyl-bonded capillary. The predominant species formed in blood when MetHb formers, such as potassium ferricyanide, hydroxylamine, sodium nitrite, and 4-dimethylaminophenol (DMAP), added at molar ratios ranging from 1:10 to 1:1 to hemoglobin, are the valency hybrid intermediates alpha3+beta2+ and alpha2+beta3+. In the detoxication of cyanide with methemoglobin, an intermediate dicyanhemimethemoglobin was demonstrated to be the predominant species in the formation of tetracyanmethemoglobin. Complex mixtures of hemoglobin derivatives were observed with DMAP at 1:1 or greater molar ratio to hemoglobin. Comparison of the MetHb values obtained with a hemoxometer indicated that the valency hybrids were measured as MetHb and the values of oxidized hemoglobin were overestimated. In cyanide poisoning, incorrect dosages of MetHb formers could be calculated, and misinterpretation of MetHb data would result from methods that fail to discriminate among the various species of MetHb.

Metabolite pharmacokinetics of soman, sarin and GF in rats and biological monitoring of exposure to toxic organophosphorus agents.

This study reports on the pharmacokinetics of the elimination of the metabolites of three toxic organophosphorus compounds (soman, sarin and GF). Urine, blood and lung tissue were collected from rats dosed subcutaneously at 75 micrograms kg-1. Urinary excretion of the metabolite was the major elimination route for these three compounds. The major differences among them were primarily the extent and rate of excretion. The hydrolyzed form, alkylmethylphosphonic acid, was the single major metabolite formed and excreted in urine by a non-saturable mechanism. Nearly total recoveries of the given doses for sarin and GF in metabolite form were obtained from the urine. The terminal elimination half-lives in urine were 3.7 +/- 0.1 and 9.9 +/- 0.8 h for sarin and GF, respectively. Soman metabolite showed a biphasic elimination curve with terminal half-lives of 18.5 +/- 2.7 and 3.6 +/- 2.2 h. Soman was excreted at a slower rate with a recovery of only 62%. Lung was the major organ of accumulation for soman. In blood the toxic agents were concentrated more in red blood cells than in plasma. The acid metabolites can serve as a better chemical marker for monitoring organophosphorus exposure in humans via their higher concentration and longer half-life in urine than the parent compounds.

Degradation of three related bis(pyridinium)aldoximes in aqueous solutions at high concentrations: examples of unexpectedly rapid amide group hydrolysis.

The principal initial degradation products of two bis(pyridinium)aldoxime organophosphate-inhibited acetylcholinesterase reactivators, 1 (HI-6) and 3 (HS-6), in concentrated nonbuffered aqueous solutions approximating potential therapeutic dosage concentrations were found to be the carboxylic acid derivatives 2 and 4 formed from the hydrolysis of the amide functional group. Compounds 2 and 4 were prepared by heating 1 and 3 in the presence of high concentrations of hydroxylamine hydrochloride and characterized by 1H and 13C NMR, IR, and UV analyses. Estimates of the rates of hydrolysis of the amide groups in 1 and 3 and in model compounds 5, 7, and 8 under similar conditions were determined. The unexpectedly rapid hydrolysis of the amide groups in 1 and 3 was attributed to both the hydrogen ion catalysis of the concentrated aqueous solutions of the unusually acidic bis(pyridinium)aldoximes 1 and 3 and general acid catalysis by the aldoxime group.

Detection of metabolites of toxic alkylmethylphosphonates in biological samples.

The major metabolites and breakdown products of some toxic organophosphonates are their respective alkymethylphosphonic acids. These acids ionize at physiological pH and are not amenable to gas chromatographic analysis in their underivatized forms. Their detection in biological samples has been difficult because of their presence at only trace levels. Existing analytical methods were developed mainly for measuring these phosphonic acids in environmental samples and at higher concentrations. In this study, we devised a gas chromatographic/mass spectrometric method to provide confirmation and quantification of the organophosphonic acids of soman (GD), sarin (GB) and GF in blood and urine. This report describes the various derivatization conditions that we have studied and demonstrates the characteristic mass spectra by different ionization techniques.

Operational evaluation of three commercial configurations of atropine/HI-6 wet/dry autoinjectors.

Commercially manufactured wet/dry autoinjectors containing atropine in solution and powdered HI-6 were evaluated using HPLC for consistency of drug delivery with various solvation times and stability of drugs postsolvation at a temperature of 40 degrees C. Three configurations of autoinjector were tested. System A (SYS A), with a specified mixing time of 5 sec, delivered a volume of 3.0 ml containing 1.86 mg of atropine sulfate and 443 mg of the bispyridinium oxime HI-6 dichloride. System B1 (SYS B1) and System B2 (SYS B2), with specified mixing times of 40 sec, delivered volumes of 2.3 ml containing 2.13 and 2.06 mg atropine citrate and 424 and 545 mg HI-6 dichloride, respectively. Average coefficients of variation for SYS A were 3.4% for atropine and 5.8% for HI-6 and for SYS B1 and B2 were 5.2% for atropine and 7.0% for HI-6 determinations. Stored from 3 to 14 days at 40 degrees C after the autoinjector contents were mixed, SYS A delivered 1.77 mg atropine sulfate and SYS B1 and B2 delivered 2.02 mg atropine citrate. The delivery of HI-6 dichloride decreased with a half-life of 34 days for SYS A, 39 days for SYS B1, and 32 days for SYS B2. This resulted in a decrease to 90% of the respective day 0 amount after 4 (SYS A) or 5 (SYS B1 or B2) days.