The identification of potential alternative biomarkers of nitrofurazone abuse in animal derived food products

Semicarbazide (SEM) was considered to be a characteristic protein-bound side-chain metabolite of the banned veterinary drug nitrofurazone and used as a marker of nitrofurazone abuse. It was recently discovered that SEM can arise in food from sources other than nitrofurazone. This uncertainty over the source of SEM may be overcome if alternative markers specific to tissue-bound nitrofurazone residues can be determined. The structure of nitrofurazone metabolites in vivo and particular proteins to which they are bound are not known. These proteins with altered structure due to the presence of the drug metabolites can be considered as potential alternative biomarkers of nitrofurazone abuse. The proteins implicated in the in vivo binding of nitrofurazone were separated and identified. A crude mixture of proteins extracted from the liver of a rat treated with the drug was separated using a series of different techniques such as preparative isoelectric focusing and size exclusion HPLC. Multiple fractions were assayed by LC-MS/MS to detect the presence of SEM. The proteins containing SEM residues were identified by peptide mass mapping using trypsin digestion and MALDI-TOF. The first protein identified as containing high concentration of SEM was albumin. It was also shown that low molecular weight species within a protein mixture whose main constituent was glutathione S-transferase contained a high concentration of SEM. The chemical composition of these components is under investigation. Preliminary data suggest the SEM forms part of a nitrofurazone metabolite conjugated to glutathione.     

2-氨基-3-苯基丙酰肼作为氨基脲敏感型胺氧化酶抑制剂的研究

目的：探讨人工合成2-氨基-3-苯基丙酰肼对氨基脲敏感型胺氧化酶（SSAO）的抑制效应。方法：根据文献合成2-氨基-3-苯基丙酰肼,通过核磁共振氢谱、红外光谱、质谱鉴定其结构。利用2-氨基-3-苯基丙酰肼对SSAO的抑制曲线,计算其IC50,结合透析实验确定2-氨基-3-苯基丙酰肼对SSAO抑制类型;建立高效液相色谱法测定2-氨基-3-苯基丙酰肼在水溶液中的浓度,验证其抑制类型。结果：2-氨基-3-苯基丙酰肼对SSAO的IC50为0.76μmol/L,透析后原液中2-氨基-3-苯基丙酰肼浓度为0.07μmol/L,其对SSAO的抑制率为78.0%。结论：2-氨基-3-苯基丙酰肼是SSAO不可逆抑制剂。     

Absence of chromosomal damage in the lymphocytes of patients treated with metronidazole for Trichomoniasis vaginalis

Chromosomal analysis was carried out on the peripheral lymphocytes of 12 patients receiving metronidazole 200 mg t.i.d. for 7 days for Trichomoniasis vaginalis infections. Samples were taken before, during and 3 weeks after completion of treatment, each patient thus acting as her own control. No increase in the chromosomal aberration frequency was found for any aberration type, either for the individual or for the group means. Thus metronidazole during short-term treatment was not found to exhibit any clastogenic effect on human lymphocytes in vivo.     

Development of a highly sensitive icELISA to detect semicarbazide based on a monoclonal antibody

A highly sensitive and specific monoclonal antibody was prepared to detect semicarbazide (SEM). Hapten 4-{[(aminocarbonyl)hydrazono]methyl}benzoic acid (4-CPSEM) was synthesised through the condensation reactions of SEM and 4-carboxybenzaldehyde (4-CBA). The active ester method was employed to couple the hapten to bovine serum albumin, keyhole limpet hemocyanin and ovalbumin (OVA) to obtain conjugates of immunogens and coating antigens. A novel hapten 2-[(aminocarbonyl)hydrazono]acetic acid (SEM-A) was also synthesised from SEM and oxoacetic acid to prepare a heterologous coating antigen (SEM-A-OVA). The linear ranges of inhibition curves in the optimised indirect competitive enzyme-linked immunosorbent assay (icELISA) method were in the range of 0.0071–0.056 ng mL^?1 and 0.018–0.209 ng mL^?1 for coating antigens SEM-A-OVA and 4-CPSEM-OVA, respectively. The IC₅₀ was 0.019 ng mL^?1 for SEM (SEM in the form of 4-nitrobenzaldehyde semicarbazone) and 0.13 ng mL^?1 for 2-nitrobenzaldehyde semicarbazone, both of which were well below the minimum required performance limit of 1 µg·kg^?1 set by the European Commission.     

Effects of the food contaminant semicarbazide following oral administration in juvenile Sprague–Dawley rats

Semicarbazide (SEM) is an azodicarbonamide by-product present in glass jar packaged foods including babyfoods, in bleaching steps and flour treatment. Experimental data showed SEM acting as osteolathyrogen agent, but few toxicological data are available in susceptible life-stages. This study aimed to evaluate effects of SEM oral administration for 28 days at 0, 40, 75, 140 mg/kg bw day during the juvenile period in Sprague–Dawley rats. Histopatological examinations of: epiphyseal cartilage – potential target of SEM lathyrogen action - testes, ovary, uterus, thyroid, thymus, spleen, adrenals, representative of the main developing organs relevant to juvenile toxicity, and neurobehavioural tests in males, were performed. Mortality at high and mid dose levels and significantly decreased body weight gain were observed in males even at the lowest dose. Lack of mineralization in cartilage at all dose levels was present. Marked alterations of spontaneous motor and exploratory behaviours were evident even at 40 mg/kg. Histological alterations were observed in all tissues; thyroid and ovary effects were present also at 40 mg/kg. The present study indicate that the NOAEL in juvenile rats is lower than 40 mg/kg for SEM oral administration. SEM administration during juvenile period exerted pleiotropic effects and further studies are suggested to elucidate mechanisms.     

Evaluation of genotoxic effects of semicarbazide on cultured human lymphocytes and rat bone marrow

Semicarbazide (SEM) belongs to the hydrazine family of chemicals, some members of which are known to possess carcinogenic potential. Information on the potential hazard of SEM itself is incomplete and the possibility that it is genotoxic cannot be ruled out. SEM is widely used as a residue marker for the banned veterinary drug nitrofurazone. Also, it occurs as a break-down product of azodicarbonamide (ADC), a chemical used as a flour treatment. Furthermore, it may form as a reaction product of hypochlorite action on food additives.     

Determination of the potency and subunit-selectivity of ribonucleotide reductase inhibitors with a recombinant-holoenzyme-based in vitro assay

Ribonucleotide reductase (RR) is an important therapeutic target for anticancer drugs. The structure of human RR features a 1:1 complex of two homodimeric subunits, hRRM1 and hRRM2. p53R2 is a newly identified homologue of hRRM2. We have devised a holoenzyme-based in vitro assay for the determination of the potency and subunit-selectivity of small-molecule inhibitors of RR. The assay was implemented using two forms of recombinant RR (hRRM2/hRRM1 and p53R2/hRRM1) and based on their [(3)H]CDP reduction activity. Hydroxyurea was used to standardize the assay. We found that the activities of hRRM2/hRRM1 and p53R2/hRRM1 were decreased by hydroxyurea in a dose-dependent manner. The -NH-OH segment of hydroxyurea was shown to be essential for inhibition. In the presence of Fe(III) and reductants, less inhibition of enzymatic activity by hydroxyurea was observed, especially for p53R2/hRRM1. The potency of four hydroxyurea analogues (Schiff bases of hydroxysemicarbazide, SB-HSC) decreased in the order SB-HSC 21 > SB-HSC 24 > SB-HSC 2 > hydroxyurea (HU) > SB-HSC 29. SB-HSC 2 and SB-HSC 24 inhibited p53R2/hRRM1 significantly more than hRRM2/hRRM1, whereas SB-HSC 21 and SB-HSC 29 showed low subunit-selectivity. Electron paramagnetic resonance (EPR) measurements showed that inhibition of RR was accompanied by reduction of its tyrosyl radical. The method was validated by comparison with data obtained using cell-based assays. We suggest that this novel recombinant-holoenzyme-based in vitro assay is a useful tool for the discovery of more potent and subunit-selective inhibitors of RR.     

Reversible time-dependent inhibition of cytochrome P450 enzymes by duloxetine and inertness of its thiophene ring towards bioactivation

Duloxetine is a selective serotonin-norepinephrine reuptake inhibitor (SNRI) approved to treat major depressive disorder and diabetic peripheral neuropathic pain. It is known to cause hepatotoxicity, in some cases leading to death. It has been reported that duloxetine causes time-dependent inhibition (TDI) of CYP1A2, CYP2B6, CYP2C19 and CYP3A4/5; but the nature of these TDI (whether reversible or irreversible) is not known. Irreversible TDI can cause clinically significant drug-drug interactions and also immune-mediated hepatotoxicity. Structurally, duloxetine possesses several toxicophores, i.e. the naphthyl and thiophene rings. It has been reported that the naphthyl ring undergoes epoxidation and was subsequently adducted to glutathione, but bioactivation related to the thiophene ring has not been completely elucidated. In this paper, the potential of duloxetine in causing irreversible TDI and generating reactive metabolites was investigated. Human liver microsomal assays demonstrated that duloxetine did not cause irreversible TDI of CYP1A2, CYP2B6, CYP2D6, CYP2C19 and CYP3A4/5. Subsequently, reactive metabolite trapping assays using soft nucleophiles (glutathione and glutathione ethyl ester) revealed a previously reported adduct at the naphthyl ring of duloxetine but not at the thiophene ring. Trapping assays utilizing a hard nucleophile (semicarbazide) did not demonstrate adducts with the thiophene ring, indicating an absence of thiophene ring opening. The hepatotoxicity of duloxetine is possibly not related to the irreversible TDI of CYP450 or the bioactivation of its thiophene moiety, but might be due to the epoxidation of its naphthyl ring.     

The dorsal periaqueductal and basolateral amygdala are necessary for the expression of conditioned place avoidance induced by semicarbazide stimulation of the dorsal periaqueductal region

The amygdala is critically involved in the regulation of unconditioned and conditioned reactions to threatening stimuli. It has been suggested that a neural circuit responsible for the production of defensive behavior elicited by the dorsal periaqueductal gray (dPAG) stimulation may project through ascending fibers to forebrain structures such as the basolateral complex of the amygdala (BLA). The present study evaluates the involvement of the dPAG and BLA in the mediation of unconditioned and conditioned responses organized in the dPAG using the open field and the conditioned place aversion (CPA) tests. In both tests, the intra-dPAG injections of semicarbazide (SEM), an inhibitor of the GABA synthesizing enzyme, was used as unconditioned stimulus (US). Using the open field test, we examine the effects of BLA inactivation with the GABA-(A) receptor agonist muscimol (MUS) on the unconditioned fear. We also investigated, through the CPA test, the effects of BLA and/or dPAG inactivation with MUS on the acquisition and the expression of the fear conditioned response. Our results showed that intra-BLA injections of MUS did not change the unconditioned fear elicited by dPAG injections of SEM. As for the CPA test, intra-BLA and intra-dPAG injections of MUS impaired the expression of CPA behavior induced by SEM injections into the dPAG. However, this inactivation of BLA did not impair the acquisition of the CPA behavior induced by injections of SEM into the dPAG. Altogether, these findings suggest that BLA does not participate in the mediation of unconditioned fear induced by dPAG chemical stimulation or in the acquisition of CPA in which aversive stimulation of the dPAG was used as US. In contrast, our results indicate that the activation of the dPAG and BLA is essential to the expression of the conditioned aversive response.     

丹皮总甙抗实验性癫痫的研究

目的：了解丹皮总甙是否具有抗小鼠实验性癫痫作用。方法：采用最大电惊厥（ＭＥＳ）及戊四唑、士的宁、氨基脲等化学性惊厥模型，观察丹皮总甙（ｔｏｔａｌｇｌｕｃｏｓｉｄｅｓｏｆｍｏｕｔａｎｃｏｒｔｅｒ，ＴＧＭ）对动物惊厥发作数、发作潜伏期及动物存活时间等指标的影响，从而分析ＴＧＭ抗惊厥作用及其时量效关系。结果：ＴＧＭ（６０、８０ｍｇ·ｋｇ－１ｉｐ；８０ｍｇ·ｋｇ－１ｉｇ）可减少小鼠ＭＥＳ发作数，其峰时为药后０．５～１ｈ；ＴＧＭ（６０～８０ｍｇ·ｋｇ－１ｉｇ）可延长戊四唑、士的宁、氨基脲所致小鼠惊厥的潜伏期及动物存活时间；同时ＴＧＭ（４０ｍｇ·ｋｇ－１ｉｐ）可增强苯巴比妥抗上述惊厥之作用。结论：ＴＧＭ（６０～８０ｍｇ·ｋｇ－１）呈剂量依赖性对抗小鼠ＭＥＳ及戊四唑、士的宁、氨基脲所致小鼠化学性惊厥，并可增强苯巴比妥抗惊厥作用。