In vivo and in vitro studies on the stereoselective hydrolysis of tri- and diglycerides by gastric and pancreatic lipases

These studies were conducted to investigate whether ascorbic acid protected guinea pigs from aflatoxin B1 (AFB1) toxicity. Young guinea pigs, fed either 0 (AA) or 25 mg (25 AA) or gavaged 300 mg ascorbic acid (300 AA) per day for 21 days, were gavaged with the LD50 dose of AFB1 on the 22nd day. Seven out of 10 animals in the AA group died within 72 hr of AFB1 administration. The livers of the animals showed regional massive necrosis and multilobular degeneration. There was no mortality in the 25 AA group. Their livers, however, showed changes similar to those seen in AA group. Serum alanine amino transferase (ALAT) and aspartate amino transferase (ASAT) levels were elevated. There was neither mortality nor pathological changes in livers in the 300 AA group. Their ALAT and ASAT levels were unaffected. In vitro production of AFM1 by liver microsomes tended to be higher than that in the other two groups. Three animals saved from the 300 AA group and continued with their supplementation were administered a second, intraperitoneal (ip) LD50 dose of AFB1 1 month after the first AFB1 dose. One animal died. Livers of the animals showed centrilobular degeneration and moderate necrosis in scattered hepatocytes. Liver microsomal cytochrome P450 and cytosolic glutathione S-transferase (GST) levels and AFM1 production were drastically reduced. ALAT and ASAT activities were raised. The results indicated that intake of 300 mg of ascorbic acid almost protected the animals from acute toxicity of AFB1 when given by gavage, but not when administered as a second dose ip.     

Chemoprevention of aflatoxin B1-induced carcinogenesis by indole-3-carbinol in rat liver--predicting the outcome using early biomarkers

Indole-3-carbinol (I3C) was examined for its ability to inhibit aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in male Fischer rats when administered either before or after the carcinogen. After 13 weeks, animals pretreated with I3C (0.5% in the diet) for 2 weeks prior to administration of AFB1 and with continuing treatment during exposure to the carcinogen were protected from development of preneoplastic lesions, as determined by the classical markers gamma-glutamyltranspeptidase (GGT) and glutathione S-transferase (GST) P. In animals receiving AFB1 for 6 weeks before treatment with I3C, there was no obvious protective effect at 13 weeks compared with animals receiving only AFB1. Using cytokeratin 18 expression as a marker, animals fed AFB1 alone had a small number of positive foci at 13 weeks. However, no cytokeratin-positive foci were visible in the majority of livers from either group receiving I3C in combination with AFB1 and after 43 weeks all animals in these groups were protected from liver tumour formation. These results suggest that expression of cytokeratin 18, a later phenotypic change in foci than induction of GST-P and GGT, correlates more closely with tumour outcome in this model. I3C appeared to retard progression of AFB1-induced carcinogenesis at both the initiation and promotion stages. Continuous treatment with I3C for 13 weeks caused significant induction of CYP1A1, 1A2, 3A and 2B1/2, GST Yc2, aflatoxin B1 aldehyde reductase and quinone reductase. Such alteration of the drug metabolizing capacity of the liver by I3C contributes to blocking of initiation, while the observed inhibition of ornithine decarboxylase, a rate limiting enzyme in polyamine biosynthesis, and of tyrosine kinase activity may contribute to the suppressive effect of I3C.     

The coffee-specific diterpenes cafestol and kahweol protect against aflatoxin B1-induced genotoxicity through a dual mechanism

The diterpenes cafestol and kahweol (C&K) have been identified in animal models as two potentially chemoprotective agents present in green and roasted coffee beans. It has been postulated that these compounds may act as blocking agents by producing a co-ordinated modulation of multiple enzymes involved in carcinogen detoxification. In this study, we investigated the effects of C&K against the covalent binding of aflatoxin B1 (AFB1) metabolites to DNA. Male Sprague-Dawley rats were treated with increasing amounts of a mixture of C&K in the diet (0-6200 p.p.m.) for 28 and 90 days. A dose-dependent inhibition of AFB1 DNA-binding was observed using S9 and microsomal subcellular fractions from C&K-treated rat liver in an in vitro binding assay. Significant inhibition was detected at 2300 p.p.m. and maximal reduction of DNA adduct formation to nearly 50% of the control value was achieved with 6200 p.p.m. of dietary C&K. Two complementary mechanisms may account for the chemopreventive action of cafestol and kahweol against aflatoxin B1 in rats. A decrease in the expression of the rat activating cytochrome P450s (CYP2C11 and CYP3A2) was observed, as well as a strong induction of the expression of the glutathione-S-transferase (GST) subunit GST Yc2, which is known to detoxify highly the most genotoxic metabolite of AFB1. These data and the previously demonstrated effects of C&K against the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis at various tissue sites suggest the potential widespread effect of these coffee components against chemical carcinogenesis.     

Effects of rumen-undegradable protein and feed intake on nitrogen balance and milk protein production in dairy cows

An experiment was designed to determine the response of milk protein production and N utilization in dairy cows to supplementation of a predominantly rumen-undegradable protein (RUP) mixture with a fixed amino acid (AA) pattern and the response to the amount of feed intake. The experiment was designed as a 6 x 6 Latin square with a 3 x 2 factorial arrangement of treatments. The factors were three concentrations of RUP supplement (4.5, 14.9, and 29.1% of dry matter intake) and two levels of feed intake restriction (10 and 20%) of the basal diet. The supplement was designed to approximate a postruminal AA pattern that was similar to bovine caseins for Met, Lys, Phe, His, and Thr. Measurements were made during the last 5 d of each 21-d period. Milk protein production responded linearly as the concentration of RUP supplement in the treatment diet increased within the given range. The difference in feed intake restriction did not affect milk protein production. Efficiency of N utilization for milk production exceeded 30% for cows fed the lowest RUP supplement. Results indicated that there is an opportunity to increase milk protein production by using RUP formulations that are balanced for AA while minimizing waste N excretion.     

Production and composition of milk from Jersey cows administered bovine somatotropin and fed ruminally protected amino acids

Eight multiparous and 4 primiparous Jersey cows averaging 92 d of lactation were utilized in a replicated 4 x 4 Latin square design with 28-d periods to determine responses to bovine somatotropin (bST) and ruminally protected Met and Lys when diets were fed that contained supplemental fat. Treatments were 1) control [no bST or ruminally protected amino acids (AA)], 2) control plus bST, 3) control plus ruminally protected AA, and 4) control plus bST plus ruminally protected AA. Dry matter intake was increased by bST but was unaffected by ruminally protected AA. Milk yield was increased by bST but was not altered by ruminally protected AA compared with the control diet. The bST tended to increase percentages of fat and total solids in milk and increased yields of fat, protein, 3.5% fat-corrected milk, and total solids. Ruminally protected AA increased percentages of fat, protein, and total solids in milk; however, yields of milk components were unaffected by ruminally protected AA. Body weight and body condition scores were unaffected by treatment. Concentrations of essential AA in plasma were unaffected by bST administration. Ruminally protected Met and Lys increased the concentration of Met and tended to increase the concentration of Lys in plasma. The lack of an increase in yields of milk and milk protein when ruminally protected AA were fed suggests that adequate amounts of Met and Lys were supplied by the control diet and protein reserves of the cows to meet the AA requirements for synthesis of milk and milk components.     

Aflatoxin B1-induced suppression of nitric oxide production in murine peritoneal macrophages

Aflatoxin B1 (AFB1), a potent hepatocarcinogen, is known to impair specific and non-specific immune responses. AFB1 mainly decreases lymphocyte functions and may also affect macrophages assisting lymphocyte functions. Macrophages play an important role in a host defense against tumors and bacteria. Furthermore, some macrophage products, including nitric oxide (NO), may be involved in cytotoxicity. The effect of aflatoxin B1 (AFB1) was investigated on NO production from murine peritoneal macrophages. Macrophages were pretreated with AFB1 for 24 h and then stimulated with lipopolysaccharide (LPS) for 24 h. AFB1 at 10 or 50 microM reduced the production of NO. Compared to vehicle control, there was a greater reduction of NO production with increased AFB1 pretreatment and LPS stimulation. AFB1 at 10 or 50 microM decreased inducible nitric oxide synthase (iNOS) activity about 24% and 28%, respectively, after stimulation with 1 microg/ml LPS and about 12% and 24%, respectively, after stimulation with 10 microg/ml LPS. AFB1 pretreatment also decreased the synthesis of iNOS protein and the mRNA of macrophages. Taken together, these results suggest that AFB1 pretreatment reduces NO production from murine peritoneal macrophages stimulated by LPS, which is mediated by the reduction of iNOS activity, mRNA, and protein.     

Medicinal herb, Thonningia sanguinea protects against aflatoxin B1 acute hepatotoxicity in Fischer 344 rats

1. Thonningia sanguinea, a plant used prophylactically against bronchial asthma in Ghana was recently found to have antioxidative and hepatoprotective actions in our laboratory. 2. In this study, the effect of T. sanguinea extract on certain biochemical indices in serum and liver of Fischer 344 rats given a single intraperitoneal (i.p.) dose (1 mg/kg) of aflatoxin B1 (AFB1) was investigated. 3. Administration of AFB1 resulted in significant increases in serum alanine aminotransferase (ALT) and glutathione S-transferase (GST) levels and a significant decrease in aniline hydroxylase activity in liver microsomes. When T. sanguinea (5 ml/kg) was intraperitoneally administered to rats 12 h and 1 h before AFB1, liver injury was significantly reduced as seen in the decreased levels of serum ALT and serum GST. However, the decrease in aniline hydroxylase activity by AFB1 was not recovered but enhanced by T. sanguinea pre-treatment. 4. Kinetic analysis of cytochrome P450 activity of rat liver microsomes in vitro demonstrated that T. sanguinea inhibited aniline hydroxylase non-competitively suggesting depression of biotransformation of AFB1 to toxic metabolites. 5. The data indicate a hepatoprotective action of T. sanguinea against AFB1-induced liver injury.     

Direct synthesis of aflatoxin B1-N7 guanine adduct: a reference standard for biological monitoring of dietary aflatoxin exposure in molecular epidemiological studies

Aflatoxin B1-N7-guanine and aflatoxin B1-human serum albumin adducts have been established as biomarkers of dietary aflatoxin exposure in epidemiological studies. Earlier chemical oxidants were used to synthesize aflatoxin B1-8,9-epoxide in vitro and its subsequent interaction with DNA or synthetic oligodeoxynucleotide was used as a source of authentic aflatoxin B1-N7-guanine adduct. In the present communication we report a simple single step procedure for the synthesis of aflatoxin B1-N7-guanine adduct using free guanine and m-chloroperbenzoic acid as the chemical oxidant for the production of AFB1-8,9-epoxide. At a molar ratio of 1:1 of AFB1-8,9-epoxide and guanine the recovery of the AFB1-N7-guanine adduct was found to be 60% while at higher molar ratios (1:2 and 1:4) of guanine the recovery of the AFB1-N7-guanine adduct was found to be low (30-40%). HPLC analysis of the AFB1-N7 guanine adduct showed a retention time identical with the retention time of the AFB1-N7-guanine adduct synthesized using calf thymus DNA. TLC-fluorodensitometric analysis indicated that the Rf of the AFB1-N7-guanine adduct was zero. Spectral analysis of the adduct synthesized showed an excitation wavelength of 360 nm and emission wavelength at 440 nm in phosphate buffer (100 mM, pH 7.4). Further, the formation of the AFB1-N7-guanine adduct was confirmed by perchloric acid treatment resulting in the destruction of the adduct. The AFB1-N7-guanine adduct thus synthesized was stable in both acidic as well as lyophilized conditions over a period of 2 weeks. The antibody capture assay showed that the antibodies produced against the antigen BSA-guanine-N7-AFB1 also cross-reacted with calf thymus DNA-AFB1 adduct, indicating specificity to the guanine-N7-AFB1 moiety. The method developed may find immediate application as a source of authentic reference standard in molecular epidemiological studies.     

Effect of aflatoxin metabolism and DNA adduct formation on hepatocellular carcinoma among chronic hepatitis B carriers in Taiwan

BACKGROUND/AIMS: Aflatoxins (AFs) are established hepatic carcinogens in several animal species. This study was performed to establish whether aflatoxin exposure may affect the risk of developing hepatocellular carcinoma in chronic hepatitis B virus carriers. METHODS: Urinary AF metabolites were measured for 43 HCC cases and 86 matched controls nested in a cohort of 7342 men in Taiwan. Thirty hepatocellular carcinoma cases and 63 controls were also tested for AFB1-albumin adducts. RESULTS: There was a dose-response relationship between urinary AFM1 levels and risk of hepatocellular carcinoma in chronic hepatitis B virus carriers. Comparing the highest with the lowest tertile of urinary AFM1 levels, the multivariate-adjusted odds ratio (OR) was 6.0 (95% confidence interval (CI) = 1.2-29.0). The hepatocellular carcinoma risk associated with AFB1 exposure was more striking among the hepatitis B virus carriers with detectable AFB1-N7-guanine adducts in urine. Compared with chronic hepatitis B virus carriers who were negative for AFB1-albumin adducts and urinary AFB1-N7-guanine, no elevated risk was observed for those who were positive for either marker. But an extremely high risk of hepatocellular carcinoma among those having both markers was found (OR = 10.0, 95% CI = 1.6-60.9). The proportion of AFB1 converted to AFM1 decreased with the progress of liver disease, whereas the formation of AFP1 increased. The difference in patterns of AFB1 metabolite formation was an independent risk factor for hepatocellular carcinoma after adjustment for total AFB1 excretion. There was a synergistic interaction between glutathione S-transferase M1 genotype and AFB1 exposure in hepatocellular carcinoma risk. CONCLUSIONS: AFB1 intake and expression of enzymes involved in AFB1 activation/detoxification may play an important role in hepatitis B virus-related hepatocarcinogenesis.     

Use of synthetic oligonucleotides for inhibition of factor NF-kappa B

The effect of the proportion of clover in the diet (200, 500 or 800 g/kg total dry matter (DM) on milk production of cows housed indoors and fed on a mixture of perennial rye-grass and white clover was measured in mid (Expt I) and late (Expt II) lactation. Higher clover contents increased the nutritive value of the diets, resulting in increased energy and protein intakes. DM intakes of cows offered 500 or 800 g clover/kg DM diets ad lib. (Expt I and Expt II, Period 1) were not significantly different but were 11-17% greater (P < 0.05) than intakes of cows fed on 200 g clover/kg total DM diets. Cows offered restricted allowances (Expt II, Period 2) had similar intakes irrespective of diet. In Expt I cows fed on 500 or 800 g clover/kg DM diets ad lib. produced 30 or 33% respectively more milk (P < 0.05) than cows fed on 200 g clover/kg total DM diets. During Expt II, Period 1, cows fed on 500 or 800 g clover/kg DM diets ad lib. produced 18 or 16% more milk (P < 0.05) respectively than cows given 200 g clover/kg total DM diets. In both these experiments the increased milk yields were due to increased intake and the higher nutritive value of the high clover diets. There was no difference in the feed conversion efficiencies of cows if maintenance energy requirements were taken into account. However, cows on restricted allowances (Expt II, Period 2) showed no significant difference in milk yield, indicating that the effect of increased nutritive value was very slight. There were no consistent effects on milk fat, protein or lactose concentrations. Concentrations of blood and milk urea increased as the clover content of the diet increased (Expt 1 only), and this was associated with increased milk non-protein N and a decreased ratio of casein N: total N. Both trials indicated an optimum clover content in the diet for milk production of 600-700 g/kg total DM.     

Immunochemical approaches to AGE-structures: characterization of anti-AGE antibodies

An extractionless method for determining aflatoxin M1 (AFM1), a major metabolite of aflatoxin B1 (AFB1), in human urine was developed. The biological fluid is injected directly into the chromatographic system after simple dilution and centrifugation. A pre-column, packed with a cation-exchange phase and coupled on-line to a column-switching liquid chromatography (LC) system, is used for sample pre-treatment and concentration. The analytes are non-selectively desorbed with the LC eluent and cleaned by means of a column-switching procedure. Pre-treatment and analysis were performed within 40 min. Average AFMI recovery reached 97% in the 10-100 ng/l range of urine. The detection limit of AFM1 in urine and milk was 2.5 ng/l for 1 ml of injected sample. A comparison with an immunoaffinity column clean-up and LC method was performed. The method was applied to determine AFM1 in the urine of AFB1 gavaged rats, and in the urine of both potentially exposed and supposedly unexposed workers. The method was also extended to milk.     

Population dynamics of the Wolbachia infection causing cytoplasmic incompatibility in Drosophila melanogaster

PURPOSE: The use of escalated radiation doses to improve local control in conformal radiotherapy of prostatic cancer is becoming the focus of many centers. There are, however, increased side effects associated with increased radiotherapy doses that are believed to be dependent on the volume of normal tissue irradiated. For this reason, accurate patient positioning, CT planning with 3D reconstruction of volumes of interest, clear definition of treatment margins and verification of treatment fields are necessary components of the quality control for these procedures. In this study electronic portal images are used to (a) evaluate the magnitude and effect of the setup errors encountered in patient positioning techniques, and (b) verify the multileaf collimator (MLC) field patterns for each of the treatment fields. METHODS AND MATERIALS: The Phase I volume, with a planning target volume (PTV) composed of the gross tumour volume (GTV) plus a 1.5 cm margin is treated conformally with a three-field plan (usually an anterior field and two lateral or oblique fields). A Phase II, with no margin around the GTV, is treated using two lateral and four oblique fields. Portal images are acquired and compared to digitally reconstructed radiographs (DRR) and/or simulator films during Phase I to assess the systematic (CT planning or simulator to treatment error) and the daily random errors. The match results from these images are used to correct for the systematic errors, if necessary, and to monitor the time trends and effectiveness of patient imobilization systems used during the Phase I treatment course. For the Phase II, portal images of an anterior and lateral field (larger than the treatment fields) matched to DRRs (or simulator images) are used to verify the isocenter position 1 week before start of Phase II. The Portal images are acquired for all the treatment fields on the first day to verify the MLC field patterns and archived for records. The final distribution of the setup errors was used to calculate modified dose-volume histograms (DVHs). This procedure was carried out on 36 prostate cancer patients, 12 with vacuum-molded (VacFix) bags for immobilization and 24 with no immobilization. RESULTS: The systematic errors can be visualized and corrected for before the doses are increased above the conventional levels. The requirement for correction of these errors (e.g., 2.5 mm AP shift) was demonstrated, using DVHs, in the observed 10% increase in rectal volume receiving at least 60 Gy. The random (daily) errors observed showed the need for patient fixation devices when treating with reduced margins. The percentage of fields with displacements of < or = 5.0 mm increased from 82 to 96% with the use of VacFix bags. The rotation of the pelvis is also minimized when the bags are used, with over 95% of the fields with rotations of < or = 2.0 degrees compared to 85% without. Currently, a combination of VacFix and thermoplastic casts is being investigated. CONCLUSION: The systematic errors can easily be identified and corrected for in the early stages of the Phase I treatment course. The time trends observed during the course of Phase I in conjunction with the isocenter verification at the start of Phase II give good prediction of the accuracy of the setup during Phase II, where visibility of identifiable structures is reduced in the small fields. The acquisition and inspection of the portal images for the small Phase I fields has been found to be an effective way of keeping a record of the MLC field patterns used. Incorporation of the distribution of the setup errors into the planning system also gives a clearer picture of how the prescribed dose was delivered. This information can be useful in dose-escalation studies in determining the relationship between the local control or morbidity rates and prescribed dose.     

The value of synthetic linear epitope analogues of La/SSB for the detection of autoantibodies to La/SSB; specificity, sensitivity and comparison of methods

Aflatoxin M1 (AFM1) is the principal hydroxylated aflatoxin metabolite present in the milk of dairy cows fed a diet contaminated with aflatoxin B1, (AFB1) and the metabolite is also present in the milk of human nursing mothers consuming foodstuffs containing the toxin. AFM1 is usually considered to be a detoxification product of AFB1 and this appears warranted if the biological endpoints involved are carcinogenicity and mutagenicity. However, it may not be a valid conclusion in the case of cytotoxicity. The metabolism of AFM1 and AFB1 have been studied in vitro using human liver microsomes. Formation of primary metabolites associated with metabolic activation to the respective epoxides reflected the differences between the carcinogenic potentials of the two toxins and, similar to AFB1, the conjugation of AFM1 epoxide with reduced GSH was catalyzed by mouse, but not human liver cytosol. Although the majority of the binding of [3H]AFB1 to microsomal protein was dependent on metabolic activation, a high level of retention of [3H]AFM1 by microsomes, nonextractable in methanol and unrelated to metabolic activation, was observed. It appears possible that this property is related to the high cytotoxicity of AFM1. Experiments using human cell line cells either expressing or not expressing human cytochrome P450 enzymes in assays of acute toxicity (MTT assays) have demonstrated a directly toxic potential of AFM1 in the absence of metabolic activation, in contrast to AFB1. Caution therefore needs to be exercised in designating the formation of AFM1 as essentially detoxification when considering a biological response in which cytotoxicity may play a significant role, e.g., immunotoxicity.     

Identification of glutathione S-transferase isozymes and gamma-glutamylcysteine synthetase as negative acute-phase proteins in rat liver

Because acute infection and inflammation affect drug metabolism and drug-metabolizing enzymes, the effect of the acute-phase response on the expression of glutathione S-transferase (GST) isoenzymes, glutathione synthesis, and several antioxidant enzymes was investigated. Hepatic expression of GST isozymes, positive and negative acute-phase reactants, and antioxidant enzymes were determined by Northern blotting and hybridization with gene-specific oligonucleotide probes after lipopolysaccharide treatment of rats. Lipopolysaccharide caused the expected acute-phase response as judged by the increased expression of positive and decreased expression of negative acute-phase proteins. The messenger RNA (mRNA) expression of the major hepatic rat GST isozymes A1, A2, A3, M1, and M2 was decreased 50% to 90%. Total hepatic GST activity toward 1-chloro-2,4-dinitrobenzene was also significantly decreased. mRNA expression of gamma-glutamylcysteine synthetase (GCS) large subunit and catalase was reduced by approximately 60%. GCS enzyme activity was also decreased, resulting in a 35% decrease in the hepatic content of reduced glutathione 4 days after lipopolysaccharide challenge. Mn-Superoxide dismutase expression was increased 13-fold, and thioredoxin level was elevated 3-fold after lipopolysaccharide challenge. The expression of all parameters determined returned to near control levels 7 days after treatment. Together, these data show that GSTs and GCS are negative acute-phase proteins and that decreased GCS activity results in a decrease in hepatic glutathione content. Thus, in addition to the phase I drug-metabolizing enzymes known to be decreased during the acute-phase response, some phase II enzymes involved in the elimination of xenobiotics and carcinogens are also decreased.     

Cloning and sequencing of the Sphingomonas (Pseudomonas) paucimobilis gene essential for the O demethylation of vanillate and syringate

A growing body of evidence supports the existence of a tissue-based renin-angiotensin system (RAS) in the vasculature, but the functional capacity of vascular RAS was not investigated in humans. In 28 normotensive healthy control subjects, the metabolism of angiotensins through vascular tissue was investigated in normal, low, and high sodium diets by the measurement of arterial-venous gradient of endogenous angiotensin (Ang) I and Ang II in two different vascular beds (forearm and leg), combined with the study of 125I-Ang I and 125I-Ang II kinetics. In normal sodium diet subjects, forearm vascular tissue extracted 36+/-6% of 125I-Ang I and 30+/-5% of 125I-Ang II and added 14.9+/-5.1 fmol x 100 mL(-1) x min(-1) of de novo formed Ang I and 6.2+/-2.8 fmol x 100 mL(-1) x min(-1) of Ang II to antecubital venous blood. Fractional conversion of 125I-Ang I through forearm vascular tissue was about 12%. Low sodium diet increased (P<.01) plasma renin activity, whereas de novo Ang I and Ang II formation by forearm vascular tissue became undetectable. Angiotensin degradation (33+/-7% for Ang I and 30+/-7% for Ang II) was unchanged, and vascular fractional conversion of 125I-Ang I decreased from 12% to 6% (P<.01). In high sodium diet subjects, plasma renin activity decreased, and de novo Ang I and Ang II formation by forearm vascular tissue increased to 22 and 14 fmol x 100 mL(-1) x min(-1), respectively (P<.01). Angiotensin degradation did not significantly change, whereas fractional conversion of 125I-Ang I increased from 12% to 20% (P<.01). Leg vascular tissue functional activities of RAS paralleled those of forearm vascular tissue both at baseline and during different sodium intake. These results provide consistent evidence for the existence of a functional tissue-based RAS in vascular tissue of humans. The opposite changes of plasma renin activity and vascular angiotensin formation indicate that vascular RAS is independent from but related to circulating RAS.     

Opiates and medial hypothalamic knife cuts cause hyperphagia through different mechanisms.

In Exp I, male Sprague-Dawley rats were given bilateral parasagittal medial hypothalamic knife cuts (KCs) or a sham procedure and fed a high-fat diet. KC and sham-operated Ss were approximately equally sensitive to the suppressive effects of naloxone (0.1–20 mg/kg, subcutaneously [sc]) on food intake. Ketocyclazocine (0.1–20 mg/kg, sc) generally increased daytime food intake in sham-operated Ss; in contrast, the normal hyperphagia of KC Ss was in most cases either unchanged or decreased by ketocyclazocine. In Exp II, neither diet composition nor hypothalamic KCs significantly affected the feeding responses to naloxone or the stimulatory effects of butorphanol tartrate (0.1–20 mg/kg, sc). It was hypothesized that the differential effects of ketocyclazocine in KC and sham-operated Ss were a consequence of the sedative effects of the drug combined with the elevated baseline of the KC Ss. This hypothesis was supported by Exp III, which showed that ketocyclazocine also reduced nocturnal intake in unoperated Ss and that butorphanol increased intake. That feeding responses to naloxone and butorphanol were essentially unchanged by hypothalamic KCs suggests that the opioid feeding system is independent of the longitudinal feeding inhibitory pathway believed to be involved in KC-induced hyperphagia. (36 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Rosemary extract and carnosol stimulate rat liver glutathione-S-transferase and quinone reductase activities

The effects of dietary intake and intraperitoneal (i.p.) administration of an extract of the spice rosemary and of the rosemary constituent carnosol on the liver activities of glutathione-S-transferase (GST) and NAD(P)H-quinone reductase (QR) in the female rat were evaluated. Rosemary extract at concentrations from 0.25 to 1.0% (by wt.) in the diet resulted in a significant 3.5- to 4.5-fold increase in liver GST and a 3.3- to 4.0-fold increase in liver QR activities compared to controls. Carnosol supplemented in the diet at levels from 0.01 to 1.0% did not enhance GST activity. When rosemary extract and carnosol were administered i.p. there was a significant increase in liver GST and QR activities. The injection of rosemary extract (200 mg/kg) was associated with 1.5-fold and 3.2-fold increases in GST and QR activities, respectively, compared to controls. The injection of carnosol at doses from 100 to 400 mg/kg was associated with 1.6- to 1.9-fold increases in GST activity and 3.1- to 4.8-fold increases in QR activity, compared to controls. These data indicate that rosemary extract in the diet or injected i.p. and carnosol administered i.p. are effective enhancers of the in vivo activity of liver GST and QR in the female rat.     

Genotoxicity of aflatoxin B1: evidence for a recombination-mediated mechanism in Saccharomyces cerevisiae

The potent liver carcinogen aflatoxin B1 (AFB1) is metabolized by cytochrome P450 to the mutagenic epoxide. We have observed that activated AFB1 also strongly induced mitotic recombination in the yeast Saccharomyces cerevisiae. To compare the recombinogenicity of AFB1 to its mutagenicity, three metabolically competent S. cerevisiae strains have been constructed. The frequencies of induced recombinants resulting from gene conversion or chromosomal translocations were determined by different prototrophic selections using two strains, whereas the inducibility of forward mutations was determined by the frequency of drug resistance in the third strain. Human cytochrome P4501A1- (CYP1A) and NADPH-cytochrome P450-oxidoreductase cDNAs were expressed in the strains to ensure intracellular metabolism to the epoxide. Exposure of the strains to AFB1 resulted in a 139- and 24-fold increase in the translocation and gene conversion frequencies, respectively, whereas the mutation frequency was increased only 3-fold. In contrast, benzo[a]pyrene-7,8-dihydrodiol and ethyl methanesulfonate induced mutation and mitotic recombination to similar degrees. We conclude that AFB1 exerted a strong recombinogenic, but only a weak mutagenic, effect. The recombinogenicity of AFB1 in yeast may indicate a mechanism for the high proportion of loss of heterozygosity that has been detected in AFB1-related human liver cancers.     

Parkinson's disease, pesticides, and glutathione transferase polymorphisms

BACKGROUND: Parkinson's disease is thought to be secondary to the presence of neurotoxins, and pesticides have been implicated as possible causative agents. Glutathione transferases (GST) metabolise xenobiotics, including pesticides. Therefore, we investigated the role of GST polymorphisms in the pathogenesis of idiopathic Parkinson's disease. METHODS: We genotyped by PCR polymorphisms in four GST classes (GSTM1, GSTT1, GSTP1, and GSTZ1) in 95 Parkinson's disease patients and 95 controls. We asked all patients for information about pesticide exposure. FINDINGS: The distribution of the GSTP1 genotypes differed significantly between patients and controls who had been exposed to pesticides (controls vs patients: AA 14 [54%] of 26 vs seven [18%] of 39; AB 11 [42%] of 26 vs 22 [56%] of 39; BB 1 [4%] of 26 vs six [15%] of 39; AC 0 vs four [10%] of 39, p=0.009). No association was found with any of the other GST polymorphisms. Pesticide exposure and a positive family history were risk factors for Parkinson's disease. INTERPRETATION: GSTP1-1, which is expressed in the blood-brain barrier, may influence response to neurotoxins and explain the susceptibility of some people to the parkinsonism-inducing effects of pesticides.     

In vitro study of the metabolic conversion of aflatoxin B1 into its epoxide

The conversion of aflatoxin B1 into its epoxide was evaluated in vitro by two different approaches based on HPLC analysis: the quantitative estimation of the tris(OH)AFB1 addition product resulting from the indirect reaction of AFB1-epoxide with tris buffer (hydroxymethyl) aminomethane and on the other hand by the quantification of the formation of the glutathione conjugate (AFB1-SG). The tris(OH)AFB1 is more sensitive than AFB1-SG in fluorescence detection. The AFB1-SG obtained (5.42 +/- 0.42 microgram) is weakly less than the quantity of tris(OH)AFB1 (6.00 +/- 0.72) obtained in the same experimental conditions.