Intramuscular prednisolone pretreatment does not influence the dermal lesions induced by topical sulfur mustard

The effect of a single intramuscular injection of prednisolone sodium phosphate (14.3 mg/kg) on the development and severity of dermal lesions induced by topical sulfur mustard was investigated in guinea pigs. Control animals received sulfur mustard alone, topical sulfur mustard and intramuscular saline, intramuscular prednisolone alone, or intramuscular saline alone. With topical sulfur mustard alone there were typical mustard lesions consisting of epidermal necrosis and hyalinization, with acute inflammation and necrosis in the dermis, the severity and extent of which were dose-related. Systemic prednisolone therapy had no influence on the nature, severity or progression of the dermal histopathologic response to topical sulfur mustard.     

芥子气诱导人真皮成纤维细胞凋亡的研究

目的：研究芥子气诱导真皮成纤维细胞凋亡的作用,探讨其治疗银屑病的作用机制。方法：采用MTT法检测不同浓度芥子气对真皮成纤维细胞增殖的抑制作用,采用AnnexinV—FITC法检测芥子气对真皮成纤维细胞凋亡指数的影响,以免疫组化法考察芥子气对肿瘤坏死因子相关凋亡诱导配体受体1（TRAILR1）表达的影响。结果：不同浓度的芥子气对真皮成纤维细胞的增殖均有抑制作用（P〈0．01）,并呈明显的剂量依赖性。真皮成纤维细胞的凋亡指数随着芥子气浓度的增加而增加。免疫组化考察结果表明,在芥子气的作用下,TRAILR1在人真皮成纤维细胞的胞膜和胞浆成强阳性表达。结论：芥子气可抑制真皮成纤维细胞的增殖,诱导其凋亡,该效应可能与芥子气促进TRAILR1的表达有关,从而可用于银屑病的治疗。     

The irreversible binding of acetylcholine mustard to muscarinic receptors in intestinal smooth muscle of the guinea-pig.

1. Acetylcholine mustard (N-2-chloroethyl-N-methyl-2-acetoxyethylamine), a potent muscarinic agonist, binds virtually irreversibly to muscarinic receptors in longitudinal muscle strips from guinea-pig small intesting, as shown by the inhibition of the binding of E13-H]-propylbenzilycholine mustard ([3-H-PrBCM), an affinity label for the muscarinin receptor. 2. A value for the apparent binding affinity of acetylcholine mustard and a value for the rate constant for the receptor alkylation reaction have been deduced from the rate of onset of the inhibition of [3-H]-PrBCM binding. 3. The kinetic constants obtained may refer largely to the interaction between acetylcholine mustard and the desensitized receptor. 4. At high concentrations acetylcholine mustard practically abolishes the contractile response to carbachol. At the concentrations acetylcholine mustard appears to have multiple actions on the tissue.     

Photosensitization of cattle in Montana: is Descurainia pinnata the culprit?

Recurrent photosensitization of cattle in Montana has been blamed on Descurainia pinnata, tansy mustard. Two feeding trials were conducted to determine if tansy mustard was phototoxic. Pen-fed cattle consumed 2.4 and 4.1 kg/hd/day of tansy mustard in the 2 trials, and no photosensitization was detected. Liver clearance of BSP was within normal limits, as were blood chemistry values for AST, CK and GGT. Field cases have confirmed that tansy mustard was present and grazed in pastures where affected animals have grazed. We suspect that other factors may be necessary to predispose cattle to photosensitization by tansy mustard, and future work will attempt to determine the cause of the photosensitization.     

Modeling for immunosuppression by sulfur mustard

The treatment and cure of patients exposed to sulfur mustard is a remaining challenge despite on-going research in this field. A severe suppression of the immune system still remains the major cause of opportunistic infections, septicemia and death in patients injured by sulfur mustard. In this report, we present a model of sulfur mustard contamination in mice, which exhibit clinical signs similar to that exhibited by patients during the Iran-Iraq war. Dose response studies were performed to determine the most appropriate dose for our model i.e., 6.35 micrograms/kg. Animals contaminated with sulfur mustard intraperitoneally showed symptoms of anorexia, diarrhea, loss of weight and blindness. Autopsy of animals showed a severe necrosis in gut and degeneration of spleen. Results shows that sulfur mustard caused an over all suppression of the immune response to sRBC as indicated by agglutination titer and DTH tests. These studies present a rodent model of sulfur mustard exposure, which can be used for further studies in this area.     

Conversion of N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard) to an aziridinium ion and its interaction with muscarinic receptors in various tissues.

A 2-chloroethylamine derivative [N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard)] of the selective muscarinic antagonist N,N-dimethyl-4-piperidinyl diphenylacetate (4-DAMP) was synthesized, and its conversion to an aziridinium ion and interaction with muscarinic receptors was investigated. When dissolved in aqueous solution at pH 7.4 and 37 degrees, 4-DAMP mustard released an equivalent amount of chloride. The release of chloride was consistent with a first-order process having a half-time of 5.7 min. The aziridinium ion reached a peak concentration at 32 min, corresponding to 75% of the initial concentration of 4-DAMP mustard. When homogenates of rat brain, heart, and submaxillary gland were incubated with 4-DAMP mustard (9 nM) for 1 hr, washed extensively, and then assayed for muscarinic receptor binding properties, a 56% decrease in the binding capacity of N-[3H]methylscopolamine in the heart and brain and a 71% decrease in the gland were observed, without a significant change in the dissociation constants. The affinity of 4-DAMP mustard and its transformation products for muscarinic receptors was determined in competitive binding experiments with N-[3H] methylscopolamine, and the results show that the aziridinium ion of 4-DAMP mustard was the most potent form, compared with the parent 2-chloroethylamine (4-DAMP mustard) and the alcoholic hydrolysis product. The rates of receptor alkylation by 4-DAMP mustard were measured in the rat heart and gland. Virtually no alkylation (less than 1%) occurred in the heart at a 4-DAMP mustard concentration of 1.6 nM, after 30 min, whereas almost 50% alkylation was observed in the gland under the same conditions. Almost complete alkylation of receptors in the gland could be achieved at a 4-DAMP mustard concentration of 200 nM, after 1 hr. Treatment of the isolated rat ileum with 4-DAMP mustard caused an irreversible blockade of contractions elicited by the muscarinic agonist oxotremorine-M, and this blockade persisted after extensive washing. The results presented here show that 4-DAMP mustard forms an aziridinium ion that binds irreversibly to muscarinic receptors and exhibits selectivity for M3, compared with M2 muscarinic receptors.     

Mercury‐induced oxidative stress in Indian mustard (Brassica juncea L.)

Mercury, a potent neurotoxin, is released to the environment in significant amounts by both natural processes and anthropogenic activities. No natural hyperaccumulator plant has been reported for mercury phytoremediation. Few studies have been conducted on the physiological responses of Indian mustard, a higher biomass plant with faster growth rates, to mercury pollution. This study investigated the phytotoxicity of mercury to Indian mustard (Brassica juncea L.) and mercury‐induced oxidative stress in order to examine the potential application of Indian mustard to mercury phytoremediation. Two common cultivars (Florida Broadleaf and Longstanding) of Indian mustard were grown hydroponically in a mercury‐spiked solution. Plant uptake, antioxidative enzymes, peroxides, and lipid peroxidation under mercury stress were investigated. Antioxidant enzymes (catalase, CAT; peroxidase, POD; and superoxide dismutase, SOD) were the most sensitive indices of mercury‐induced oxidative response of Indian mustard plants. Indian mustard effectively generated an enzymatic antioxidant defense system (especially CAT) to scavenge H₂O₂, resulting in lower H₂O₂ in shoots with higher mercury concentrations. These two cultivars of Indian mustard demonstrated an efficient metabolic defense and adaptation system to mercury‐induced oxidative stress. A majority of Hg was accumulated in the roots and low translocations of Hg from roots to shoots were found in two cultivars of Indian mustard. Thus Indian mustard might be a potential candidate plant for phytofiltration/phytostabilization of mercury contaminated waters and wastewater. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2009.     

葡磷酰胺在大鼠体内的代谢研究

目的初步阐明葡磷酰胺在大鼠体内的代谢情况。方法大鼠静脉给予葡磷酰胺50 mg·kg^-1，采用液相色谱-质谱联用法对大鼠尿中的代谢产物进行分析。分别在正、负离子两种检测方式采用一级全扫描、产物离子扫描、中性丢失扫描、母离子扫描等多种扫描方式下对代谢物进行检测。结果在正离子检测方式下，除原形外共检测到两种代谢物，即异磷酰胺氮芥和异磷酰胺氮芥脱卤素后生成的单胺丙啶基衍生物；在负离子检测方式下，由于异磷酰胺氮芥等化合物无质谱响应而只检测到葡磷酰胺原形。稳定性实验证明在大鼠尿中所检测到的两种化合物为代谢产物而非降解产物。结论葡磷酰胺在大鼠尿样中主要以原形的形式存在，另外还检测到异磷酰胺氮芥和异磷酰胺氮芥的单胺丙啶基衍生物两种代谢产物。     

Altered expression of lung cytochrome P450 3A1 in rat after exposure to sulfur mustard

Expression of cytochromes P450 (CYP) and glutathione S-transferases in the lung may be affected by inhaled pollutants. We have investigated the effect of sulfur mustard on the expression of CYP 1A1, 2B1, 2E1 and 3A1, as well as of alpha-, micro- and pi-glutathione S-transferases in rat lung. Sulfur mustard (0.025, 0.05 or 0.1 mg/kg) or its vehicle was administered to anaesthetized animals by intratracheal injection. Expression of CYP and glutathione S-transferases was analysed 24 hr after administration of the vesicant warfare using western blotting. Preservation of airway epithelium integrity after animal exposure to sulfur mustard was confirmed by histological examination of tracheal and lung tissues from control and treated animals. Constitutive levels of CYP 2B1 and 3A1 proteins were found in lung tissue from control rats, whereas CYP 1A1 and 2E1 proteins were not detected. Animal exposure to sulfur mustard enhanced CYP 3A1 protein levels by 80 to 103%. In contrast, exposure to sulfur mustard neither modified CYP 2B1 expression, nor led to detectable expression of CYP 1A1 or 2E1. Constitutive levels of alpha-, micro- and pi-glutathione S-transferase proteins were found in lung tissue from control rats. Exposure to sulfur mustard had no effect on expression of either of the glutathione S-transferases. Our results show that intratracheal exposure to sulfur mustard selectively increases CYP 3A1 expression in rat lung. Taking into account the major role of CYP of the 3A family in the metabolism of drugs, up-regulation of CYP 3A1 by sulfur mustard might have important therapeutic consequences.     

Half-life of oxazaphosphorines in biological fluids

Plasma AUC and half-life values for cyclophosphamide were determined in rats manipulated to hydroxylate cyclophosphamide at different rates; plasma AUC and apparent half-life values for two pharmacologically important metabolites of cyclophosphamide, viz. 4-hydroxycyclophosphamide/aldophosphamide and phosphoramide mustard, were also determined in these animals. Apparent plasma half-life values for 4-hydroxycyclophosphamide/aldophosphamide and phosphoramide mustard increased with an increase in plasma half-life values for cyclophosphamide. AUC values for cyclophosphamide increased approximately linearly with an increase in its plasma half-life but AUC values for 4-hydroxycyclophosphamide/aldophosphamide and phosphoramide mustard remained approximately constant with an increase in their respective apparent plasma half-life values. Given that the cytotoxic effects of cyclophosphamide are directly proportional to AUC values for 4-hydroxycyclophosphamide/aldophosphamide and/or phosphoramide mustard, we conclude that changes in the rate of cyclophosphamide hydroxylation will not alter the systemic toxic and therapeutic responses to a given dose of cyclophosphamide. Actual half-life values for 4-hydroxycyclophosphamide/aldophosphamide and phosphoramide mustard after the iv infusion of these agents were also determined. A comparison of the actual plasma half-life values for cyclophosphamide (29 min), 4-hydroxycyclophosphamide/aldophosphamide (14 min), and phosphoramide mustard (14 min) with the apparent plasma half-life values obtained for 4-hydroxycyclophosphamide/aldophosphamide (34 min) and phosphoramide mustard (55 min) following cyclophosphamide administration suggests that the major determinant with regard to the apparent plasma half-life of 4-hydroxycyclophosphamide/aldophosphamide is its rate of formation whereas in the case of phosphoramide mustard, an additional determinant, perhaps efflux from the cell, is operative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancing effects of mustard oil on preneoplastic hepatic foci development in Wistar rats

Dietary habits are known to be the major contributory factor in the development of cancer. Mustard oil, which is extensively used in India and elsewhere as a flying and cooking medium, is reported to induce an inflammatory response. The development of altered hepatic foci is an early carcinogenic change in rat liver in diethylnitrosamine (DEN)-induced hepatocarcinogenesis. In the present study, the development of preneoplastic lesions was observed following administration of mustard oil (0.5 mL/day for 8 weeks) in DEN-initiated and partially hepatomized Wistar rats. A significant decrease in the relative and absolute liver weight of mustard oil-exposed rats was recorded. The results revealed a significant increase in the number and area of placental glutathione-S-transferase (GST-P) and gamma-glutamyl transpeptidase (GGT)-positive foci in mustard oil-administered animals. The GST-P- and GGT-positive foci were more prominent in the animals given boiled (up to 300 degrees C for 3 hours) mustard oil in comparison to the animals given fresh mustard oil. These results indicate the possible tumourigenic risk associated with mustard oil consumption.     

芥子气诱导真皮成纤维细胞分泌肿瘤坏死因子相关凋亡诱导配体的时效和量效研究

目的探讨芥子气诱导真皮成纤维细胞分泌肿瘤坏死因子相关凋亡诱导配体（TRAIL）的规律。方法采用酶联免疫吸附（ELISA）方法,检测不同浓度的芥子气调节TRAIL分泌的量效和时效关系。结果真皮成纤维细胞自身可分泌一定量的TRAIL,芥子气作用后,可显著上调其TRAIL的分泌（P〈0．05）,并呈时间依赖性和剂量依赖性。结论芥子气治疗银屑病的作用机制可能与促进细胞分泌TRAIL有关。     

Changes in the oxidative stress/anti-oxidant system after exposure to sulfur mustard and antioxidant strategies in the therapy,a review

Sulfur mustard, in a chemical name bis(2-chloroethyl) sulfide, is a chemical warfare agent. It is cytotoxic and blister forming once spread over the skin. Though exact molecular mechanism of sulfur mustard toxic action remains unknown, inflammation and oxidative stress development are considered as the most relevant pathological consequences. Applications of either low-molecular weight antioxidants or cofactors for enzymatic antioxidants are considered as suitable ways how to ameliorate the poisoning. In this article, survey of literature on countermeasures against sulfur mustard poisoning are given and evidence of oxidative stress role during sulfur mustard poisoning and availability of antioxidants for the therapy are discussed.     

Chemopreventive effects of mustard (Brassica compestris) on chemically induced tumorigenesis in murine forestomach and uterine cervix

As there is a strong correlation between diet and cancer, the dietary constituents that inhibit mutagenesis and/or carcinogenesis are of paramount importance for the prevention of human cancer. In the present study, cancer chemopreventive potentials of different doses of mustard (Brassica compestris) seed mixed diets were evaluated against benzo(a)pyrene [B(a)P]-induced forestomach tumorigenesis and 3-methylcholantrene (MCA)-induced uterine cervix tumorigenesis. Results showed a significant inhibition of stomach tumour burden (tumours/ mouse) by mustard seeds. Tumour burden was 7.08 +/- 2.47 in the B(a)P-treated control group, whereas it was reduced to 1.36 +/- 1.12 (P<0.001) by the 2.5% dose and 1.18 +/- 0.87 (P<0.001) by the 5% dose of mustard seeds. The cervical carcinoma incidence, as compared to MCA-treated control group (73.33%), was reduced to nil (P<0.05) by the 5% diet of mustard seeds and to 13.33% (P<0.05) by the 7.5% diet of mustard seeds. The effect of the 2.5% and 5% mustard seed mixed diets was also examined on the antioxidant enzymes, glutathione content, lactate dehydrogenase (LDH) and lipid peroxidation in the liver of Swiss albino mice. The glutathione-S-transferase-specific activity was increased (P<0.05) by the 2.5% dose, whereas there was no significant change in the activity of DT-diaphorase. In antioxidant systems, significant elevation of the specific activities of superoxide dismutase and catalase was observed with both doses of mustard seeds (P<0.05). The level of reduced glutathione (GSH) measured as nonprotein sulphydryl content was elevated by the 2.5% dose of mustard seeds only (P<0.05). Lipid peroxidation measured as formation of thiobarbituric acid reactive substances production showed significant inhibition (P<0.05) by the 5% dose of mustard seed mixed diet. LDH activity was decreased significantly (P<0.05) by both the doses. The results strongly suggest the cancer chemopreventive potentials of mustard seeds and their ability to enhance the antioxidant defence system and in turn provide protection against the toxic effects of carcinogens. It is likely that the use of mustard seeds in the diet may contribute to reducing the risk of cancer incidence and burden in the human population.     

Correlations of alkylating activity and mutagenicity in bacteria of cytostatic drugs

Alkylating activity of cytostatic drugs was studied in relation to their mutagenicity and toxicity in E. coli WP2 uvrA. Four classes of directly acting cytostatic drugs were studied: nitrogen mustards (nitrogen mustard, melphalan, chlorambucil and phosphoramide mustard, a metabolite of cyclophosphamide), ethyleneimine derivatives (Thio-TEPA, TEPA and triethylenemelamine), busulfan, and halogenated nitrosoureas. The reference compounds included methyl methanesulfonate, ethyleneimine and methylnitrosourea. Guanosine alkylation was determined by fluorometry. The rate of guanosine and nitrobenzylpyridine alkylation agreed well. Nitrogen mustard derivatives and triethylenemelamine were the most potent alkylating agents among the cytostatic drugs; nitrogen mustard was 5 to 10 times more active than methyl methanesulfonate. Ethyleneimine derivatives, busulfan and the nitrosoureas were relatively weak alkylating agents. Nitrogen mustard and triethylenemelamine were the most potent mutagens to bacteria; they were also among the most toxic drugs studied.     

Analysis of thiodiglycol in urine of victims of an alleged attack with mustard gas

A procedure for the semi-quantitative determination of thiodiglycol, a metabolite of the vesicant mustard gas, in urine has been developed. Thiodiglycol was converted into mustard gas using concentrated HCl at temperatures close to 100 degrees C. The headspace of the solution containing mustard gas, was trapped on an adsorption tube filled with Tenax-GC which was subsequently analyzed by gas chromatography/mass spectrometry. Using 10 mL of urine, a detection limit of a few ng/mL of thiodiglycol was achieved. The procedure was applied to urine samples obtained from Iranian patients who were the alleged victims of an attack by chemical warfare agents (probably mustard gas). A number of control samples were investigated as well. Thiodiglycol was found in the urine of the Iranian patients in concentrations varying between 3 and 140 ng/mL. However, the detection of thiodiglycol in concentrations up to 55 ng/mL in control samples excluded the unambiguous verification of the use of mustard gas against the Iranian patients.     

Chemically stable, lipophilic prodrugs of phosphoramide mustard as potential anticancer agents

Benzyl phosphoramide mustard (3), 2,4-difluorobenzyl phosphoramide mustard (4), and methyl phosphoramide mustard (5) were examined as lipophilic, chemically stable prodrugs of phosphoramide mustard (2). These phosphorodiamidic esters are designed to undergo biotransformation by hepatic microsomal enzymes to produce 2. The rate of formation of alkylating species, viz., 2, from these prodrugs and their in vitro cytotoxicity toward mouse embryo Balb/c 3T3 cells were comparable to or better than that of cyclophosphamide (1). Preliminary antitumor screening against L1210 leukemia in mice, however, suggests that these prodrugs are devoid of any significant antitumor activity in vivo.     

Alkylation of guanosine by phosphoramide mustard, chloromethine hydrochloride and chlorambucil

Guanosine was reacted in vitro with phosphoramide mustard, chloromethine hydrochloride, and chlorambucil. The products were isolated by HPLC and characterized by UV and fluorescence spectroscopy, and C-8 tritium exchange. The primary products were 7-alkylguanosines according to such evidence. Phosphoramide mustard had 1/10 of the apparent alkylation activity of two other mustards. The primary 7-alkylguanosines were unstable at pH 7.4 and 37 degrees; t1/2 were 3 min. for chloromethine hydrochloride, 2.7 hrs for chlorambucil and 3.0 hrs for phosphoramide mustard. Both dechlorination at the unbound arm of the mustard and imidazole ring opening og guanosine appeared to account for such instability.     

A RAPID METHOD FOR QUANTITATING THE EXTENT OF DNA DAMAGE INDUCED BY SULFUR MUSTARD IN HUMAN LYMPHOCYTES FOR DRUG SCREENING

We previously characterized the effects of sulfur mustard on deoxyribonucleic acid (DNA) patterns in exposed human lymphocytes and demonstrated how poly (ADP-ribose) polymerase inhibitors (PARPI) alter these effects. These studies were conducted utilizing labor-intensive and time-consuming DNA isolation andgelelectrophoresis procedures.Toconduct mechanistic studiesandto screen antivesicant therapeutic regimens for their capacity to block or alter the DNAdamaging effects of sulfur mustard, a faster and less labor-intensive method was developed. Preparations of human lymphocytes, isolated from the blood of normal volunteers, were exposed to sulfur mustard (1 × 10 -8 M to 1 × 10 -3 M) andincubatedat 37 o C for 0-24h.The effects of sulfur mustard on the DNA of the lymphocytes were determinedusing flow cytometry by measuring theincrease in the fluorescence of the DNA peak caused by the uptake of propidium iodide (PI) by the fragmented DNA. The increase in the fluorescence of the DNA depended on both the concentration of sulfur mustard to which the cells were exposed and the lengthof time following exposure to sulfur mustard. Anincrease inthe binding of PI to the sulfur mustard-exposed lymphocyte DNA is detected as early as 1hpostexposure.Theincrease inPIfluorescencerose sharplyduring thefirst 4 h and then appeared to increase linearly between 4 and 24 h after sulfur mustard exposure. This method results in a more rapid and less labor-intensive determination of DNA damage, enabling kinetic and mechanistic determination of DNA damage.     

Analysis for plasma protein biomarkers following an accidental human exposure to sulfur mustard

Following an accidental human exposure to a vesicating agent, plasma samples were analyzed for specific biomarkers of sulfur mustard. One individual suffered chemical burns over 6.5% of the body surface area and required hospitalization; the second individual developed a single, small blister. Plasma specimens from both individuals were examined using two different assays. The first assay targeted sulfur mustard adducts to cysteine-34 of albumin using affinity chromatography, enzyme digestion, and analysis of the alkylated peptide fragment using liquid chromatography-tandem mass spectrometry. The second assay targeted alkylation sites of glutamic and aspartic acids of plasma proteins. Following precipitation of plasma proteins, the sulfur mustard adducts were cleaved from the protein using base, derivatized, and analyzed using gas chromatography-mass spectrometry. Samples obtained over a 42-day period from the individual requiring hospitalization produced positive results for sulfur mustard adducts using both assays. Observed levels of the sulfur mustard biomarker decreased by approximately 75% between days 2 and 42 for both assays. Samples obtained over a six-day period from the individual with a single, small blister produced positive results for the albumin adduct assay. Observed levels were much lower than levels from the hospitalized patient. Blood samples from suspected human exposures to sulfur mustard have only rarely been made available for analysis by sensitive and specific laboratory assays. The data presented here add significantly to the small database of information that currently exists on human biomarkers of sulfur mustard exposure, linking a well-documented exposure event with levels of plasma protein adducts.