Toxic effects of 2-methyl-4-dimethylaminoazobenzene in normal and partially hepatectomized rats

In order to obtain a more precise definition of the conditions under which 2-methyl-4-dimethylaminoazobenzene (2-Me-DAB) and liver cell proliferation play a role in the initiation of hepatocarcinogenesis, the toxicity of 2-Me-DAB for normal and partially hepatectomized rats was investigated. Continuous feeding of a basal low protein, low riboflavin diet supplemented with 2-Me-DAB was found to be highly toxic for male albino rats. All animals fed on such a diet died before 200 days. Sham operation and partial hepatectomy (PH) at 30 days of 2-Me-DAB feeding reduced the median survival time from 122 days to 107 and 94 days, respectively. Transfer to the basal diet after 30 days of 2-Me-DAB feeding and PH prolonged the median survival time to 216 days while 97% of the rats returned to the normal complete diet after the same treatments survived for more than 300 days. 2-Me-DAB was not necrogenic and there was no evidence of reparative proliferation or hepatic tumor formation in any group. Feeding rats with the 2-Me-DAB containing diet for 1 month delayed and strongly inhibited the mitotic response of the liver to the stimulus of partial hepatectomy. This is the result of a blockage of the cells in G1 as revealed by the fact that only 1% of the hepatocytes became labeled when 2-Me-DAB fed animals were injected with tritiated thymidine prior to sacrifice at 24 h post-hepatectomy, as compared to 40% in rats fed the normal or the control basal diet. This inhibitory effect of 2-Me-DAB is reversible however since rats returned to the normal diet for 1 or 2 months after 2-Me-DAB feeding showed percentages of mitoses and labeling indices comparable to those of control animals following PH. The number of abnormal mitoses was high (13%) in regenerating livers of rats fed 2-Me-DAB and the lesions responsible for this effect are apparently not repaired since 2-Me-DAB fed rats partially hepatectomized after being transferred to the normal diet for 1 or 2 months showed the same number of mitotic irregularities. The present results suggest that assays with 2-Me-DAB as 'pure initiator' or agent of selective toxicity should be pursued in attempts to improve existing experimental models of hepatocarcinogenesis.     

Detecting cadmium during ultrastructural characterization of hepatotoxicity

BackgroundThe threat of cadmium (Cd), which is the cause of itai-itai disease in Japan, is still complicated and confusing, especially for digestive system, such as liver disease. One of the most keys of this problem is demonstrating that the hepatotoxicity is indeed induced by Cd. Therefore, we attempt detecting Cd at microscale during ultrastructural imaging of liver tissue.Methods12 rats were divided randomly into two experimental groups: control and Cd-treated. Treated rats were intraperitoneal injected with 1 mg/kg body weight cadmium chloride (CdCl₂) for 4 weeks (5 P.M each day for 6 days/week). At the end of the exposure period, liver tissue samples were processed into ultrathin sections for analysis of advanced analytical transmission electron microscopy and X-ray energy dispersive spectroscopy (TEM/X-EDS) investigations. Ultrastructural images and X-ray energy dispersive spectrum were acquired at microscale.ResultsCd can cause changes in the structure of the organelle, including the collapse of the membrane structure in the cell, the destruction of the internal structure of the organelle, the mitochondrial swelling, the expansion of the endoplasmic reticulum, and the appearance of inclusions. Cadmium bioaccumulation is detected in the mitochondria at microscale by TEM/X-EDS, which is the visual evidence of morphological changes of mitochondria related to Cd.ConclusionThe combination of detailed ultrastructure and microscale X-ray energy dispersive spectroscopy (X-EDS) characterization of cadmium hepatotoxicity demonstrate that cadmium indeed leads to mitochondrial damage, which is helpful for further investigation of the pathological mechanism of cadmium hepatotoxicity.     

白花鬼针草中多烯炔类成分的分离及其生物活性研究

为了解白花鬼针草(Bidens pilosa var.radiata)的化学成分,采用多种色谱技术从其提取物中分离多烯炔类成分,并对其生物活性进行研究。结果表明,从白花鬼针草乙酸乙酯提取部位中分离鉴定出4个多烯炔类化合物,分别为5-acetoxy-2-phenylethinyl-thiophene (1)、1-phenylhepta-1,3,5-triyne (2)、5-phenyl-2-(1-propynyl)-thiophene (3)和icthyothereol acetate (4)。体外活性评价结果表明,化合物1～4均具有中等抗MRSA活性,且均对人肝LO_2细胞无毒性。首次为化合物1提供核磁数据并进行结构解析,化合物3、4为首次从该属植物中分离得到。     

The role of proteomics in toxicology: identification of biomarkers of toxicity by protein expression analysis

Proteomics, i.e. the high throughput separation, display and identification of proteins, has the potential to be a powerful tool in drug development. It could increase the predictability of early drug development and identify non-invasive biomarkers of toxicity or efficacy. This review provides an introduction to modern proteomics, with particular reference to applications in toxicology. A literature search was carried out to identify studies in two broad classes: screening/predictive toxicology, and mechanistic toxicology. The strengths and limitations of current methods and the likely impact of techniques in drug development are also considered. Proteomics can increase the speed and sensitivity of toxicological screening by identifying protein markers of toxicity. Proteomics studies have already provided insights into the mechanisms of action of a wide range of substances, from metals to peroxisome proliferators. Current limitations involving speed of throughput are being overcome by increasing automation and the development of new techniques. The isotope-coded affinity tag (ICAT) method appears particularly promising. The application of proteomics to drug development has given rise to the new field of pharmacoproteomics. New associations between proteins and toxicopathological effects are constantly being identified, and major progress is on the horizon as we move into the post-genomic era.     

Effect of Cassia fistula Linn. leaf extract on diethylnitrosamine induced hepatic injury in rats

The hepatoprotective and antioxidant effect of Cassia fistula Linn. leaf extract on liver injury induced by diethylnitrosamine (DEN) was investigated. Wistar rats weighing 200+/-10g were administered a single dose of DEN (200mg/kg b.w., i.p.) and left for 30 days. For hepatoprotective studies, ethanolic leaf extract (ELE) of C. fistula Linn. (500mg/kg b.w., p.o.) was administered daily for 30 days. AST, ALT, ALP, LDH, gamma-GT and bilirubin were estimated in serum and liver tissue. Lipid peroxidation (LPO), SOD and CAT were also estimated in liver tissue as markers of oxidative stress. DEN induced hepatotoxicity in all the treated animals were evident by elevated serum ALT, AST, ALP and bilirubin levels and a simultaneous fall in their levels in the liver tissue after 30 days. Induction of oxidative stress in the liver was evidenced by increased LPO and fall in the activities of SOD and CAT. ELE administration for 30 days prevented the DEN induced hepatic injury and oxidative stress. In conclusion, it was observed that ELE of C. fistula Linn. protects the liver against DEN induced hepatic injury in rats.     

Discovery of novel indole-based aroylhydrazones as anticonvulsants: Pharmacophore-based design

A number of novel melatonin derivatives, containing aroylhydrazone moieties, were synthesized and explored in vivo for anticonvulsant activity, neurotoxicity in ICR mice as well as in-vitro for cytoxicity and oxidative stress in rats. The structures and configurations were confirmed by NMR, FTIR, HRMS and crystal X-ray diffraction method. For selection of potent structures for synthesis a pharmacophore model was used. Two compounds 3e, with a 2-furyl moiety fragment and 3f with 2-thienyl fragment, showed a potency in maximal electroshock (MES) test (ED₅₀ = 50.98 mg kg⁻¹, PI > 5.88 and ED₅₀ = 108.7 mg kg⁻¹; PI > 2.76), respectively, higher than melatonin (ED₅₀ = 160.3 mg kg⁻¹, PI > 1.87). The compounds 3c, 3e, 3f and 3i suppressed psychomotor seizures in the 6 Hz test and 3c was the most potent with higher ED₅₀ = 13.98 mg kg⁻¹ and PI of > 21.46 compared to that of melatonin (ED₅₀ = 49.76 mg kg⁻¹ and PI of > 6.03) in mice. None of the compounds displayed neurotoxicity in the rota-rod test. The novel melatonin derivatives exerted weak cytotoxic effects while 3f showed the lowest hepatoxic effects comparable to that of the positive control melatonin in rats. The high affinities to the elucidated pharmacophore model of the novel melatonin compounds derived from the inclusion of aroylhydrazone moiety in the indole scaffold yielded suitable candidates with anticonvulsant activity in the MES and 6 Hz test of psychomotor seizures.     

alpha-Tocopherol protects against alpha-naphthylisothiocyanate-induced hepatotoxicity in rats less effectively than melatonin

The protective effect of alpha-tocopherol (alpha-Toc), which exerts antioxidant and anti-inflammatory actions, against alpha-naphthylisothiocyanate (ANIT)-induced hepatotoxicity in rats was compared with that of melatonin because orally administered melatonin is known to protect against ANIT-induced hepatotoxicity in rats through its antioxidant and anti-inflammatory actions. Rats intoxicated once with ANIT (75 mg/kg, intraperitoneal (i.p.)) showed liver cell damage and biliary cell damage with cholestasis at 24 h, but not 12 h, after intoxication. ANIT-intoxicated rats received alpha-Toc (100 or 250 mg/kg) or melatonin (100 mg/kg) orally at 12 h after intoxication. The alpha-Toc administration protected against liver cell damage in ANIT-intoxicated rats, while the melatonin administration protected against both liver cell damage and biliary cell damage with cholestasis. ANIT-intoxicated rats had increased hepatic lipid peroxide concentration and myeloperoxidase activity at 12 and 24 h after intoxication. ANIT-intoxicated rats also had increased serum alpha-Toc and non-esterified fatty acid (NEFA) concentrations at 12 and 24 h after intoxication and increased serum triglyceride and total cholesterol concentrations at 24h. The administration of alpha-Toc to ANIT-intoxicated rats increased the hepatic alpha-Toc concentration with further increase in the serum alpha-Toc concentration and attenuated the increased hepatic lipid peroxide concentration and myeloperoxidase activity and serum NEFA concentration at 24 h after intoxication. The melatonin administration did not affect the hepatic alpha-Toc concentration but attenuated the increased hepatic lipid peroxide concentration and myeloperoxidase activity and serum alpha-Toc, NEFA, triglyceride, and total cholesterol concentrations at 24 h after ANIT intoxication. These results indicate that orally administered alpha-Toc protects against ANIT-induced hepatotoxicity in rats possibly through its antioxidant and anti-inflammatory actions less effectively than orally administered melatonin.     

Acetaminophen-induced hepatotoxicity of gel entrapped rat hepatocytes in hollow fibers

An important application of primary hepatocyte cultures is for hepatotoxicity research. In this paper, gel entrapment culture of rat hepatocytes in miniaturized BAL system were evaluated as a potential in vitro model for hepatotoxicity studies in comparison to monolayer cultures. After exposure for 24 and 48 h to acetaminophen (2.5 mM), gel entrapped hepatocytes were more severely damaged than hepatocyte monolayer detected by methyl thiazolyl tetrazolium (MTT) reduction, intracellular glutathione (GSH) content, reactive oxygen species (ROS) levels, urea genesis and albumin synthesis. CYP 2E1 activities detected by 4-nitrocatechol (4-NC) formation were higher in gel entrapped hepatocytes than in hepatocyte monolayers while the addition of CYP 2E1 inhibitor, diethyl-dithiocarbamate (DDC), more significantly reduced acetaminophen-induced toxicity in gel entrapped hepatocytes. In addition, protective effects of GSH, liquorice extract and glycyrrhizic acid against acetaminophen hepatotoxicity were clearly observed in gel entrapped hepatocytes but not in hepatocyte monolayer at an incubation time of 48 h. Overall, gel entrapped hepatocytes showed higher sensitivities to acetaminophen-induced hepatotoxicity than hepatocyte monolayer by a mechanism that higher CYP 2E1 activities of gel entrapped hepatocytes could induce more severe acetaminophen toxicity. This indicates that gel entrapped hepatocytes in hollow fiber system could be a promising model for toxicological study in vitro.