Insecticide interaction with carrier and neuroproteins.

Binding of alpha-, beta-, gamma-hexachlorocyclohexane, p,p'-DDT and p,p'-DDE with bovine serum albumin (BSA) and locust brain homogenate was studied. Binding affinities of pesticides were higher for the locust brain homogenates than for BSA. Results of uptake by isolated locust brain revealed higher uptake of gamma-HCH than alpha-HCH. gamma-HCH uptake was also higher from locust haemolymph than either from BSA or from buffer.     

Dietary hexachlorocyclohexane induced changes in blood and liver lipids in albino mice.

Dietary intake of technical hexachlorocyclohexane (HCH) by albino mice for 2 weeks (at 400 and 800 ppm) resulted in hyperlipemia. Significant increase in triglycerides, phospholipids and cholesterol fractions of blood was observed in these animals. Dietary intake of gamma-isomer of HCH for 2 weeks (at 200 ppm) did not have any effect on blood lipid profile, but at 400 ppm level produced higher contents of phospholipids and cholesterol. The hepatomegaly produced by dietary technical HCH or gamma-HCH was not accompanied by fatty metamorphosis of liver. Hypertriglyceridemia caused by HCH was accompanied by lower triglyceride levels in liver, suggesting a possible higher rate of secretion from liver.     

Biochemical toxicity of hexachlorocyclohexane and its gamma-isomer in albino mice

Dietary hexachlorocyclohexane (HCH) and gamma-isomer of HCH produced significant increase in liver weights of mice. Elevated levels of alanine and aspartate aminotransferases and of alkaline phosphatase in the blood of these animals suggested hepatotoxicity. Hepatic soluble enzymes--aspartate aminotransferase and lactate dehydrogenase--were markedly lowered. Among the hepatic lysosomal enzymes, acid phosphatase and acid cathepsin were increased in the experimental animals. Hepatic glucose-6-phosphatase was lowered by HCH while aldolase activity was increased. Hydrolytic enzymes in small intestine, viz., disaccharidases, lipase, amylase, dipeptidase and phosphatases, were also affected by dietary HCH and gamma-HCH. The results suggested cellular toxicity in hepatocytes of HCH and gamma-HCH fed animals, and also interference in gastrointestinal absorption.     

Lindane-induced biochemical perturbations in rat serum and attenuation by omega-3 and Nigella sativa seed oil

Lindane (gamma-hexachlorocyclohexane, gamma-HCH), a highly persistent organochlorine insecticide is neurotoxic at acute doses and has been reported to induce oxidative stress in cells and tissues. In this study, we investigated the antioxidant property of Nigella sativa seed oil (N.O) and omega-3 polyunsaturated fatty acids (omega3) against gamma-HCH-induced oxidative hepatic and renal damage in male rats serum. Rats were orally given sublethal dose of gamma-HCH (12 mg/kg, 24 h prior to decapitation), while N.O (0.3 ml/kg) and omega3 (20 mg/kg) were given every 48 h for 20 days single or together, or also combined with gamma-HCH. gamma-HCH caused a significant increase in the levels of serum total lipids, cholesterol, and triglycerides by 49, 61 and 30% respectively, while HDL-cholesterol decreased by 45% compared to control group. Pretreatment with omega3 and N.O prior gamma-HCH administration re-established the altered biochemical features and alleviated the harmful effects of gamma-HCH on lipid profile. The concentration of serum total protein and albumin was significantly decreased by 35 and 45% respectively in rats treated with gamma-HCH compared to control. gamma-HCH also caused hepatic and renal damage, as observed from the elevated serum levels of urea, creatinine, total bilirubin and uric acid contents and aminotransferases (AST and ALT), phosphatases (ACP and ALP) and lactate dehydrogenase (LDH) activities. Co-administration of omega3 and N.O reversed the hazardous effects induced by gamma-HCH on the liver and kidney and also protected acetylcholinesterase from the inhibitory action of gamma-HCH as well as suppressed the lipid peroxidation. Thus, the results show that omega3 and N.O might prevent oxidative stress and attenuate the changes in the biochemical parameters induced by gamma-HCH in male rats.     

Complete nucleotide sequence of an exogenously isolated plasmid, pLB1, involved in gamma-hexachlorocyclohexane degradation

The alpha-proteobacterial strain Sphingobium japonicum UT26 utilizes a highly chlorinated pesticide, gamma-hexachlorocyclohexane (gamma-HCH), as a sole source of carbon and energy, and haloalkane dehalogenase LinB catalyzes the second step of gamma-HCH degradation in UT26. Functional complementation of a linB mutant of UT26, UT26DB, was performed by the exogenous plasmid isolation technique using HCH-contaminated soil, leading to our successful identification of a plasmid, pLB1, carrying the linB gene. Complete sequencing analysis of pLB1, with a size of 65,998 bp, revealed that it carries (i) 50 totally annotated coding sequences, (ii) an IS6100 composite transposon containing two copies of linB, and (iii) potential genes for replication, maintenance, and conjugative transfer with low levels of similarity to other homologues. A minireplicon assay demonstrated that a 2-kb region containing the predicted repA gene and its upstream region of pLB1 functions as an autonomously replicating unit in UT26. Furthermore, pLB1 was conjugally transferred from UT26DB to other alpha-proteobacterial strains but not to any of the beta- or gamma-proteobacterial strains examined to date. These results suggest that this exogenously isolated novel plasmid contributes to the dissemination of at least some genes for gamma-HCH degradation in the natural environment. To the best of our knowledge, this is the first detailed report of a plasmid involved in gamma-HCH degradation.     

Degradation of beta-Hexachlorocyclohexane by Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26

Beta-Hexachlorocyclohexane (beta-HCH) is the most recalcitrant among the alpha-, beta-, gamma-, and delta-isomers of HCH and causes serious environmental pollution problems. We demonstrate here that the haloalkane dehalogenase LinB, reported earlier to mediate the second step in the degradation of gamma-HCH in Sphingomonas paucimobilis UT26, metabolizes beta-HCH to produce 2,3,4,5,6-pentachlorocyclohexanol.     

Mice with a deletion in the gene for CCAAT/enhancer-binding protein beta have an attenuated response to cAMP and impaired carbohydrate metabolism

Fifty percent of the mice homozygous for a deletion in the gene for CCAAT/enhancer-binding protein beta (C/EBP beta-/- mice; B phenotype) die within 1 to 2 h after birth of hypoglycemia. They do not mobilize their hepatic glycogen or induce the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK). Administration of cAMP resulted in mobilization of glycogen, induction of PEPCK mRNA, and a normal blood glucose; these mice survived beyond 2 h postpartum. Adult C/EBP beta-/- mice (A phenotype) also had difficulty in maintaining blood glucose levels during starvation. Fasting these mice for 16 or 30 h resulted in lower levels of hepatic PEPCK mRNA, blood glucose, beta-hydroxybutyrate, blood urea nitrogen, and gluconeogenesis when compared with control mice. The concentration of hepatic cAMP in these mice was 50% of controls, but injection of theophylline, together with glucagon, resulted in a normal cAMP levels. Agonists (glucagon, epinephrine, and isoproterenol) and other effectors of activation of adenylyl cyclase were the same in liver membranes isolated from C/EBP beta-/- mice and littermates. The hepatic activity of cAMP-dependent protein kinase was 80% of wild type mice. There was a 79% increase in the concentration of RI alpha and 27% increase in RII alpha in the particulate fraction of the livers of C/EBP beta-/- mice relative to wild type mice, with no change in the catalytic subunit (C alpha). Thus, a 45% increase in hepatic cAMP (relative to the wild type) would be required in C/EBP beta-/- mice to activate protein kinase A by 50%. In addition, the total activity of phosphodiesterase in the livers of C/EBP beta-/- mice, as well as the concentration of mRNA for phosphodiesterase 3A (PDE3A) and PDE3B was approximately 25% higher than in control animals, suggesting accelerated degradation of cAMP. C/EBP beta influences the regulation of carbohydrate metabolism by altering the level of hepatic cAMP and the activity of protein kinase A.     

Growth inhibition by metabolites of gamma-hexachlorocyclohexane in Sphingobium japonicum UT26

The growth of a gamma-hexachlorocyclohexane (gamma-HCH)-degrading bacterium Sphingobium japonicum (formerly Sphingomonas paucimobilis) UT26 in rich medium was inhibited by gamma-HCH. This growth inhibition was not observed in a mutant that lacked the initial or second step enzymatic activity for gamma-HCH degradation, suggesting that metabolites of gamma-HCH are toxic to UT26. Two metabolites of gamma-HCH, 2,5-dichlorophenol (2,5-DCP) and 2,5-dichlorohydroquinone (2,5-DCHQ), showed a direct toxic effect on UT26 and other sphingomonad strains. Because only 2,5-DCP accumulated during gamma-HCH degradation, 2,5-DCP is thought to be a main compound for growth inhibition.     

Metabolism of the 16-androstene steroids in primary cultured porcine hepatocytes

The hepatic metabolism of the 16-androstene steroids was investigated using isolated porcine hepatocytes. This study demonstrated that the liver is capable of producing both phase I and phase II steroid metabolites from 16-androstene steroid precursors. 16-Androstene metabolites were recovered by solid-phase extraction and identified by gas chromatography-mass spectrometry (GC-MS). When 5alpha-androstenone was provided as a substrate, both 3beta- and 3alpha-androstenol were produced as well as a metabolite that showed evidence of hydroxylation. Incubations with the various 16-androstene steroids produced metabolic profiles which suggested that the major role of the liver is phase II conjugation. Sulfoconjugated 16-androstene steroids included androstadienol, 5alpha-androstenone, 3beta-, 3alpha-androstenol, and possibly the hydroxylated metabolite of 5alpha-androstenone. It was determined that hydroxysteroid sulfotransferase (HST) is the likely candidate for the sulfoconjugation of the 16-androstene steroids within the liver. Despite the capacity of the hepatocytes to sulfoconjugate the 16-androstene steroids, the principle metabolites produced from incubations with 5alpha-androstenone, 3beta-, and 3alpha-androstenol were glucuronide conjugates, accounting for approximately 68% of all phase II metabolism. These findings underline the importance of steroid conjugation and suggest that hepatic metabolism of the 16-androstene steroids may influence the levels of 5alpha-androstenone present in the circulation, and thus, capable of accumulating in fat.     

Comparative genome analysis with the human genome reveals chicken genes associated with fatness and body weight

The selection of meat-type chickens (broilers) for rapid growth has been accompanied by excessive fat deposition. In this study, we analysed 53 candidate genes that are associated with obesity and obesity-related traits in humans, for which we found chicken orthologues by BLAST searches. We have identified single nucleotide polymorphisms (SNPs) with significant differences in allele frequencies between broilers and layers in each of the following six candidate genes: adrenergic, beta-2-, receptor, surface (ADRB2); melanocortin 5 receptor (MC5R); leptin receptor (LEPR), McKusick-Kaufman syndrome (MKKS), milk fat globule-EGF factor 8 protein (MFGE8) and adenylate kinase 1 (AK1). To examine associations with fatness and/or body weight, we used birds of extreme phenotypes in F(2) and backcross populations with varying levels of abdominal fat weight per cent (%AFW) and body weight. We then assessed the level of gene expression by real-time PCR. In two genes, ADRB2 and MFGE8, we found significant association with %AFW. The ADRB2 gene was found to have a significantly higher expression in the liver of lean chickens compared with those of the fat individuals. We believe that this approach can be applied for the identification of other quantitative genes.     

Frequency of non-alcoholic fatty liver disease in overweight/obese children and adults: Clinical,sonographic picture and biochemical assessment

Non-alcoholic fatty liver disease (NAFLD) is a condition defined by significant lipid accumulation (5–10%) in hepatic tissue in the absence of significant chronic alcohol consumption. We aim to detect frequency of fatty liver among overweight/obese adults and children and associated clinical; anthropological measures; biochemical; genetic and imaging studies. Eighty three consecutive adults and 72 children included in the study. All patients underwent clinical measurements of height, body weight, body mass index (BMI), waist and hip circumference. Biochemical investigations were done to all subjects including liver function tests; lipid profile; fasting blood glucose; insulin resistance (IR); high sensitivity C reactive protein (hs-CRP); adiponectin and genotyping of adiponectin genes. Abdominal ultrasonography was done to search for fatty liver; to measure subcutaneous fat thickness (SFT) and visceral fat thickness (VFT). Fatty liver was detected in 47 (65.3%) children and in 52 (62.7%) adults. Correlation analysis in both groups revealed that enlarged liver was highly positively correlated to age; BMI, systolic blood pressure (SBP), diastolic blood pressure (DBP); waist circumference; hip circumference, subcutaneous fat thickness (SFT) and Visceral fat thickness (VFT), alanine aminotransferase (ALT), aspartate aminotransferase/alanine aminotransferase (AST/ALT). In addition in adults to fasting blood glucose, cholesterol, triglycerides (TG), low density lipoprotein (LDL), IR and hs-CRP. Homozygous T adiponectin genotype at position +276 was significantly increased among children with enlarged liver size and hs-CRP. NAFLD affects a substantial portion of adults and children; it is associated with the metabolic syndrome.     

A novel pathway for the biodegradation of gamma-hexachlorocyclohexane by a Xanthomonas sp. strain ICH12

AIM: To isolate gamma-hexachlorocyclohexane (HCH)-degrading bacteria from contaminated soil and characterize the metabolites formed and the genes involved in the degradation pathway. METHODS AND RESULTS: A bacterial strain Xanthomonas sp. ICH12, capable of biodegrading gamma- HCH was isolated from HCH-contaminated soil. DNA-colony hybridization method was employed to detect bacterial populations containing specific gene sequences of the gamma-HCH degradation pathway. linA (dehydrodehalogenase), linB (hydrolytic dehalogenase) and linC (dehydrogenase) from a Sphingomonas paucimobilis UT26, reportedly possessing gamma-HCH degradation activity, were used as gene probes against isolated colonies. The isolate was found to grow and utilize gamma-HCH as the sole carbon and energy source. The 16S ribosomal RNA gene sequence of the isolate resulted in its identification as a Xanthomonas species, and we designated it as strain ICH12. During the degradation of gamma-HCH by ICH12, formation of two intermediates, gamma-2,3,4,5,6-pentachlorocyclohexene (gamma-PCCH), and 2,5-dichlorobenzoquinone (2,5-DCBQ), were identified by gas chromatography-mass spectrometric (GC-MS) analysis. While gamma-PCCH was reported previously, 2,5-dichlorohydroquinone was a novel metabolite from HCH degradation. CONCLUSIONS: A Xanthomonas sp. for gamma-HCH degradation from a contaminated soil was isolated. gamma-HCH was utilized as sole source of carbon and energy, and the degradation proceeds by successive dechlorination. Two degradation products gamma-PCCH and 2,5-DCBQ were characterized, and the latter metabolite was not known in contrasts with the previous studies. The present work, for the first time, demonstrates the potential of a Xanthomonas species to degrade a recalcitrant and widespread pollutant like gamma-HCH. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the isolation and characterization of a novel HCH-degrading bacterium. Further results provide an insight into the novel degradation pathway which may exist in diverse HCH-degrading bacteria in contaminated soils leading to bioremediation of gamma-HCH.     

裂殖壶藻藻油DHA对高脂饮食诱导肥胖小鼠的影响

【目的】肥胖症是一种慢性代谢类疾病,具有较高的发病率和高危后果。研究表明,n-3多不饱和脂肪酸(n-3 Polyunsaturated fatty acids,n-3 PUFAs),特别是二十二碳六烯酸(Docosahexaenoic acid,DHA)对与肥胖症相关疾病有较好的防治效果,对体内脂质代谢有重要的调节作用。探讨裂殖壶藻(Schizochytrium sp.)藻油DHA对高脂饮食诱导肥胖小鼠体重、脂肪组织重量、血脂、肝和脂肪组织病理形态和脂质代谢相关基因表达的影响。【方法】通过高脂饮食建立小鼠肥胖模型,以体重增幅15%为标准分出肥胖小鼠。试验共分五组:(1)低脂对照组;(2)高脂模型组;(3)高脂+低剂量藻油组(50 mg DHA/kg);(4)高脂+中剂量藻油组(100 mg DHA/kg);(5)高脂+高剂量藻油组(200 mg DHA/kg)。其中,藻油处理组灌服相应剂量藻油,低脂对照组和高脂模型组灌胃同等体积玉米油。处理9周后,腹腔麻醉,摘眼球取血并分离血清,测血清中甘油三酯、胆固醇和高密度脂蛋白含量;之后处死小鼠,分离附睾、肾周和肠系膜脂肪组织及肝脏,称湿重;附睾脂肪和肝组织切片进行HE染色,观察病理变化情况;利用RT-PCR检测附睾脂肪组织中激素敏感脂酶(Hormone sensitive lipase,HSL)基因的m RNA表达情况。【结果】藻油处理组小鼠体重没有显著下降,但是腹部脂肪重量显著降低、脂肪细胞体积明显小于高脂模型组;同时血清中甘油三酯、胆固醇含量显著降低,肝组织异位脂肪堆积明显减少;脂肪组织中HSL基因的表达水平显著提高。【结论】裂殖壶藻藻油DHA处理能显著降低高脂饮食导致的小鼠腹部脂肪积累并改善血脂,可能有利于肥胖症的防治。     

Sex-differential expression of metabolism-related genes in response to a high-fat diet

Objective: The aim of this work was to determine the sex‐associated differences in the expression of genes related to lipid metabolism and fuel partitioning in response to a high‐fat (HF) diet in rats, and whether this is linked to the higher tendency of males to suffer from metabolic disorders. Methods and Procedures: Male and female Wistar rats were fed for 6 months on a normal‐fat (NF) or an HF diet. Body weight, fat depot weight, lipid concentration in liver, blood metabolites, and the expression of genes involved in fuel metabolism and partitioning in the liver, white adipose tissue (WAT), and skeletal muscle were measured. Results: Female rats fed on an HF diet gained more weight and had a greater increase in the adiposity index than male rats, while the circulating insulin levels remained unaltered; these animals also showed an increased expression of genes related to the energy influx in WAT and with fat utilization in skeletal muscle. Male but not female rats showed increased hepatic peroxisome proliferator–activated receptor‐ α (PPAR‐ α ) and CPT1L mRNA expression, suggesting enhanced lipid handling and oxidation by this organ, and have a higher triacylglycerol content in liver. Male rats under the HF diet also displayed higher blood insulin levels. Discussion: These results show sex‐dependent differences in lipid handling and partitioning between tissues in response to an HF diet, with females showing a higher capacity for storing fat in adipose tissue and for oxidizing fatty acids in muscle. These adaptations can help to explain the lower tendency of females to suffer from obesity‐linked disorders under the conditions of an HF diet.     

Synthesis and characterization of 5-azido-UDP-glucuronic acid. A new photoaffinity probe for UDP-glucuronic acid-utilizing proteins.

A new active site-directed photoaffinity analogue, [beta-32P]5-azido-UDP-glucuronic acid (UDP-GlcA), was enzymatically synthesized from [beta-32P]5-N3UDP-Glc using UDP-glucose dehydrogenase. The product was characterized by its mobility on ion exchange and two thin-layer chromatographic systems, by its UV absorbance at 288 nm, and the loss of this absorbance after UV irradiation of the compound. Photoincorporation of [beta-32P]5-N3UDP-GlcA into bovine liver UDP-Glc dehydrogenase (EC 1.1.1.22) was saturable with an apparent Kd of 12.5 microM, and was inhibited by the known active-site effectors UDP-GlcA, UDP-Glc, and UDP-xylose. When human liver microsomes with known UDP-glucuronosyltransferase (EC 2.4.1.17) activities were photolabeled with [beta-32P]5-N3UDP-GlcA, major photolabeled bands of 35-37 and 50-54 kDa were detected. When rat liver microsomes from phenobarbital-injected rats were photolabeled with [beta-32P]5-N3UDP-GlcA, there was a marked increase in photoincorporation of a 51-kDa protein as compared with control animals. Evidence is presented which suggests that the photolabeled 51-54-kDa proteins in the liver microsomes from both tissues are UDP-glucuronosyltransferase and that [beta-32P]5-N3UDP-GlcA represents a new alternative approach in the study of UDP-glucuronosyltransferase and other UDP-GlcA-utilizing enzymes.     

Lipid oxidation in plasma and tissues from estrogenized chicken hens

Four groups of 5-month-old chicken hens were given estradiol treatments and/or 5% dietary oil supplement for 14 days, after which blood plasma, liver, heart, and skeletal muscle were analyzed for lipid oxidation by TBA assay for malonaldehyde. Plasma from estradiol-treated birds had 8-fold higher levels of malonaldehyde compared to untreated birds. The bulk of this effect was due to a 5-fold increase in plasma lipid, but this lipid also contained a 70% higher concentration of malonaldehyde. Estradiol treatments produced significantly increased TBA numbers in liver, heart, and skeletal muscle. Corn oil supplementation significantly increased the malonaldehyde concentration in fat extracted from liver and heart, but not from plasma or skeletal muscle. It was concluded that estradiol treatment, in addition to generally increasing the deposition of fat in plasma and organs, also enhanced the concentration of malonaldehyde equivalents in plasma and organ fat.     

Isolation and characterization of a novel gamma-hexachlorocyclohexane-degrading bacterium.

The natural biotic capacity of soils to degrade gamma-hexachlorocyclohexane (gamma-HCH, lindane) was estimated using an enrichment technique based on the ability of soil bacteria to develop on synthetic media and degrade the xenobiotic compound, used as the sole source of carbon and energy. Bacterial inocula from relatively highly contaminated soils (from wood treatment factories) were found to promote efficiently the degradation of gamma-HCH, which subsequently permitted isolation of a competent gamma-HCH-degrading microorganism. The decrease of gamma-HCH concurrently with the release of chloride ions and the production of CO2 demonstrated the complete mineralization of gamma-HCH mediated by the isolate. This was confirmed by gas chromatography-mass spectrometry analyses showing that degradation subproducts of gamma-HCH included an unidentified tetrachlorinated compound and subsequently 1,2,4-trichlorobenzene and 2,5-dichlorophenol. The two linA- and linB-like genes coding, respectively, for a gamma-HCH dehydrochlorinase and a dehalogenase were characterized by using a PCR strategy based on sequence homologies with previously published sequences from Sphingomonas paucimobilis UT26. Nucleotide sequence analysis of the linA-like region revealed the presence of a 472-bp open reading frame exhibiting high homology with the linA gene from S. paucimobilis, while a preliminary study also indicated strong homology among the two linB genes. All enzymes involved in the gamma-HCH degradative pathway appear to be extracellular and encoded by genes located on the chromosome, although numerous cryptic plasmids have been detected.     

Fish oil supplementation in F1 generation associated with naproxen, clenbuterol, and insulin administration reduce tumor growth and cachexia in Walker 256 tumor-bearing rats

Weanling female Wistar rats were supplemented with fish oil (1 g/kg body weight) for one generation. The male offspring received the same supplementation until to adult age. Rats supplemented with coconut fat were used as reference. Some rats were inoculated subcutaneously with a suspension (2 x 10(7) cells/mL) of Walker 256 tumor. At day 3, when the tumor was palpable, rats were treated with naproxen (N) (0.1 mg/mL), clenbuterol (Cb) (0.15 mg/kg body weight), and insulin (I) (10 U/kg body weight). At day 14 after tumor inoculation, the animals were killed. Tumor was removed and weighed. Blood, liver, and skeletal muscles were also collected for measurements of metabolites and insulin. In both tumor-bearing untreated rats and tumor-bearing rats supplemented with coconut fat, tumor growth, triacylglycerol, and blood lactate levels were higher, and glycogen content of the liver, blood glucose, cholesterol and HDL-cholesterol levels were lower as compared with the non-tumor-bearing and fish oil supplemented groups. Fish oil supplementation of tumor-bearing rats led to a partial recovery of the glycogen content in the liver and a full reversion of blood glucose, lactate, cholesterol, and HDL-cholesterol levels. The treatment with N plus Cb plus I attenuated cancer cachexia and decreased tumor growth in both coconut fat and fish oil supplemented rats. In conclusion, chronic fish oil supplementation decreased tumor growth and partially recovered cachexia. This beneficial effect of fish oil supplementation was potentiated by treatment with naproxen plus clenbuterol plus insulin.