Diffusional kurtosis imaging of the developing brain.

BACKGROUND AND PURPOSE: Diffusional kurtosis imaging is an extension of DTI but includes non-Gaussian diffusion effects, allowing more comprehensive characterization of microstructural changes during brain development. Our purpose was to use diffusional kurtosis imaging to measure age-related microstructural changes in both the WM and GM of the developing human brain. MATERIALS AND METHODS: Diffusional kurtosis imaging was performed in 59 subjects ranging from birth to 4 years 7 months of age. Diffusion metrics, fractional anisotropy, and mean kurtosis were collected from VOIs within multiple WM and GM structures and subsequently analyzed with respect to age. Diffusional kurtosis tractography images at various stages of development were also generated. RESULTS: Fractional anisotropy and mean kurtosis both showed age-related increases in all WM regions, reflecting progression of diffusional anisotropy throughout development, predominantly in the first 2 years of life (eg, 70% and 157% increase in fractional anisotropy and mean kurtosis, respectively, from birth to 2 years for the splenium). However, mean kurtosis detected continued microstructural changes in WM past the fractional anisotropy plateau, accounting for more delayed isotropic changes (eg, 90% of maximum fractional anisotropy was reached at 5 months, whereas 90% of maximum mean kurtosis occurred at 18 months for the external capsule). Mean kurtosis may also provide greater characterization of GM maturation (eg, the putamen showed no change in fractional anisotropy but an 81% change in mean kurtosis from birth to 4 years 7 months). CONCLUSIONS: Mean kurtosis detects significant microstructural changes consistent with known patterns of brain maturation. In comparison with fractional anisotropy, mean kurtosis may offer a more comprehensive evaluation of age-related microstructural changes in both WM and GM and is potentially a valuable technique for studying brain development.

The effect of modulation of glutathione levels on markers for aflatoxin B1-induced cell damage.

The modulatory effect of glutathione levels on markers for aflatoxin B1 (AFB1)-induced cell damage has been investigated in the rat (susceptible specie) and the (mouse resistant specie). The concentration of GSH was depleted and increased by administering paracetamol (PAM) and cysteine respectively and activities ofglutathione S-transferase (GST) and gamma-glutamyl transpeptidase (gamma-GT) were determined. The effect ofAFB1 on hepatic lipid peroxidation in both species was also investigated. Treatment of rats with 2 mg/kg.bwt AFB1 intraperitoneally caused a depletion of GSH in the liver to a minimum at 6 h (80% of the control value) and the level returned to normal after 24 h. GST was similarly increased to a maximum at 6 h and the level also returned to normal after 24 h. GSH and GST activities were not significantly affected in AFB1-treated mice. Orally administered PAM (400 mg/kg.bwt) caused a depletion of GSH with a minimum at 6 h (59% and 36% of the control rats and mice respectively). Pretreatment of AFB1 with PAM produced a serious depletion at 6 h (34% and 35% of the control rats and mice respectively). GST activities were also marginally increased in both animals. AFB1 pretreatment mediated (P < 0.001) hepatic lipid peroxidation in rats but not in mice as assessed by the formation of thiobarbituric acid reactive substances (TBARS). Treatment of rats and mice with oral cysteine (50 mg/kg bwt) elicited a significant elevation of GSH. Administration of cysteine with AFB to rats attenuated the toxic effects of AFB1 on GSH and inhibited the formation of TBARS. gamma-GT activity was significantly increased when AFB1 alone was administered to rats but was not increased (P > 0.05) when cysteine was pretreated alone with AFB1. Combined treatment of AFB, and PAM induced 177% increase in gamma-GT activity. Overall, our results suggest that the metabolism of aflatoxin B, by GSH does not lead to the formation of toxic products but rather GSH plays a protective role in AFB1-induced cell damage and GSH pathway is less utilised in mice.

Repression of cellular anaplerosis as the hypothesized mechanism of gamma-linolenic acid-induced toxicity to tumor cells.

In in vitro cultures, the cell is virtually isolated and can no longer rely on mechanisms for physiological regulation of substrate availability found in tissues. More emphasis is laid on utilization of preponderant substrate in a proposed reciprocal relationship between glycolysis and free fatty acid (FFA) oxidation for energy. Supraphysiological concentrations of gamma-linolenic acid and some other polyunsaturated fatty acids (PUFAs) therefore suppress glycolysis but also inhibit FFA oxidation initiated through a cytochrome P450-mediated epoxidation of PUFA to inhibit fatty acid synthase (FAS) activity. FAS inhibition accumulates malonyl CoA which inhibits carnitine palmitoyl transferase I and prevents FFA oxidation. The cell is starved of energy and anabolic intermediates, leading to decreased proliferation or death for tumor cells. Tumor cells are more vulnerable to this induced toxicity due to possession of specific phenotypes of elevated expression for FAS and pyruvate kinase, type M2, both factors inducing tumor cell apoptosis on inhibition.

Peroxisome proliferator-activated receptor- gamma expression in human malignant and normal brain, breast and prostate-derived cells.

The constitutive and gamma -linolenic acid (GLA)-induced expression of peroxisome proliferator-activated receptor gamma (PPAR gamma) immunoreactive protein in a panel of human malignant brain (U87MG, T98G); breast (MCF-7, MB MDA-231, MB MDA 435) and prostate (ALVA, DU-145, LNCaP, PC3) cell lines have been compared with those for their normal cell counterparts, the human normal astrocyte (NHA), mammary epithelial (HMEC) and prostate epithelial (PrEC) cells, respectively. Constitutive levels of expression for PPAR gamma protein were significantly higher in the malignant cell lines relative to their normal cells. GLA supplementation did not affect the protein expression in malignant cells but caused 6- and 3-fold increases in normal breast and prostate cells, respectively. Since activation of PPAR gamma protein in some human malignant cell lines has been demonstrated to induce tumour cell death, these findings signal the need to exploit the significantly elevated expression of this protein in the therapy of human cancer.

Identification of a fatty acid delta6-desaturase deficiency in human skin fibroblasts.

Polyunsaturated fatty acid (PUFA) utilization was investigated in skin fibroblasts cultured from a female patient with an inherited abnormality in lipid metabolism. These deficient human skin fibroblasts (DF) converted 85;-95% less [1-14C]linoleic acid (18:2n-6) to arachidonic acid (20:4n-6), 95% less [3-14C]tetracosatetraenoic acid (24:4n-6) to docosapentaenoic acid (22:5n-6), and 95% less [1-14C]-linolenic acid (18:3n-3) and [3-14C]tetracosapentaenoic acid (24:5n-3) to docosahexaenoic acid (22:6n-3) than did normal human skin fibroblasts (NF). The only product formed by the DF cultures from [1-14C]tetradecadienoic acid (14:2n-6) was 18:2n-6. However, they produced 50;-90% as much 20:4n-6 as the NF cultures from [1-14C]hexadecatrienoic acid (16:3n-6), [1-14C]gamma-linolenic acid (18:3n-6), and [1-14C]dihomo-gamma-linolenic acid (20:3n-6), PUFA substrates that contain Delta6 double bonds. DF also contained 80% more 18:2n-6 and 25% less 20:4n-6. These results suggested that DF are deficient in Delta6 desaturation. This was confirmed by Northern blots demonstrating an 81;-94% decrease in Delta6-desaturase mRNA content in the DF cultures, whereas the Delta5-desaturase mRNA content was reduced by only 14%. This is the first inherited abnormality in human PUFA metabolism shown to be associated with a Delta6-desaturase deficiency. Furthermore, the finding that the 18- and 24-carbon substrates are equally affected suggests that a single enzyme carries out both Delta6 desaturation reactions in human PUFA metabolism.

Inhibition of aflatoxin B1 genotoxicity in human liver-derived HepG2 cells by kolaviron biflavonoids and molecular mechanisms of action.

Kolaviron biflavonoids have demonstrated antihepatotoxic activity in animal studies. The present study investigated the possible chemopreventive potential of kolaviron in inhibiting aflatoxin B1 (AFB1) genotoxicity in HepG2 cells. Kolaviron inhibition of AFB1-induced cytotoxicity by clonogenic assay and genotoxicity by [3H]thymidine incorporation in unscheduled DNA synthesis were evaluated, including antioxidant potential of kolaviron determined by its reduction in the intracellular reactive oxygen species level induced by hydrogen peroxide. Induction of AFB1-detoxicating enzymes such as cytochrome P450 3A4 (3A4) and glutathione S-transferases (GSTs) A1-1/ A2-2 (alpha) and M1B (mu) was determined by reverse transcription polymerase chain reaction (RT-PCR) and northern blotting for the messages and western immunoblot analysis for protein. Kolaviron significantly (P < 0.01) and dose-dependently inhibited the cytotoxicity (by 71.6%) and genotoxicity (47.1%) of AFB1 in HepG2 cells. The antioxidant potential of kolaviron compared favourably with values for the standard antioxidant trolox C (53.8% at only 4.5 x 10(-2)-fold kolaviron concentration) but was below that of butylated hydroxyanisole (58.1% at a ninefold kolaviron concentration). It induced about threefold increases in the messages for 3A4 and GSTs alpha and mu, including a twofold increase in GSTalpha protein. Kolaviron may have chemopreventive potential in inhibition of human AFB1 genotoxicity and possibly hepatocarcinogenesis.

Induction of cyclooxygenase-2 by overexpression of the human catalase gene in cerebral microvascular endothelial cells.

Prostaglandin (PG) formation by the inducible (type 2) cyclooxygenase (COX-2) and reactive oxygen species (ROS) have been proposed to play important roles in cerebrovascular pathological processes. To explore the relationship between ROS and COX-2 expression, adenovirus (Ad) vectors containing cDNA for human antioxidant enzymes including catalase (AdCAT:), copper/zinc superoxide dismutase (AdCu/ZnSOD), and manganese superoxide dismutase (AdMnSOD) were transferred into murine cerebral microvascular endothelial cells. AdCAT: (100 multiplicity of infection) infection increased the content and enzymatic activity of cellular Cat threefold and decreased the intracellular peroxide level. The expression of COX-2 mRNA and protein in cell lysates was up-regulated, and the amount of PGE(2) formed from exogenous arachidonic acid increased following AdCAT: infection in a dose-dependent manner, paralleling the expression of COX-2 protein. The AdCAT:-induced increase in PGE(2) formation was inhibited by NS-398, a selective inhibitor of COX-2 enzymatic activity. AdCAT: infection did not change the expression of the constitutive (type 1) COX protein. Although AdCu/ZnSOD and AdMnSOD infection increased the expression of superoxide dismutase proteins, COX-2 expression was not induced. An in vitro nuclear transcription assay indicated that overexpression of the Cat gene increases the transcription of the COX-2 gene. Furthermore, the stability of COX-2 mRNA induced by lipopolysaccharide was increased after AdCAT: gene transfer. These results indicate that AdCAT: gene transfer induces the transcriptional activation of the COX-2 gene and increases COX-2 mRNA stability. Therefore, peroxide may have regulatory effect on COX-2 function in the cerebral microcirculation.

Chloramphenicol and ampicillin-induced changes in rat hepatic esterase and amidase activities.

The influence of ampicillin and chloramphenicol administered intraperitoneally singly or in combination on the protein content and the activities of hepatic esterase and amidase have been investigated in rats. The results have been compared to the effects of phenobarbitone (inducer) and p-nitrophenyl-phosphate (inhibitor) of hepatic hydrolases. Ampicillin pretreatment reduced protein level and amidase activity by 3.5% each but caused a significant increase (8.1%) in total esterase activity compared to controls. Chloramphenicol treatment caused an overall decrease in protein level, esterase and amidase activities respectively by 11%, 11%, and 35% over controls. Combined administration of both drugs resulted in a decrease in protein, esterase and amidase activities by 11.5%, 12.5%, and 41.2% respectively, thus mimicking the effects obtained with chloramphenicol alone. The changes induced by administration of the drugs particularly in combination on the constituent enzymes of rat hepatic hydrolases may affect the ability of the body to deal with exposure to environmental chemicals if extrapolated to man.

Chemoprevention of 2-acetylaminofluorene-induced hepatotoxicity and lipid peroxidation in rats by kolaviron--a Garcinia kola seed extract.

The effect of kolaviron, a mixture of Garcinia biflavonoid 1 (GB1), Garcinia biflavonoid 2 (GB2) and kolaflavanone, used in the treatment of various ailments in southern Nigeria on hepatotoxicity and lipid peroxidation induced by 2-acetylaminofluorene (2-AAF) in rats was investigated. The ability of butylated hydroxyanisole (BHA) to attenuate the toxic effect of 2-AAF was also examined. Kolaviron administered orally to rats at a dose of 100mg/kg body weight twice a day for 1 week before challenge with 2-AAF (200mg/kg feed) and continuously for 3 weeks at a single dose of 200mg/kg body weight reversed the 2-AAF-mediated decrease in final body weight and relative organ weights, especially the liver. BHA was administered at a dose of 7.5g/kg feed to the animals for 4 weeks. The extract decreased significantly the 2-AAF-mediated increase in the activity of aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase and ornithine carbamyl transferase by 58%, 62%, 60% and 67%, respectively. BHA elicited respectively 55%, 63%, 57% and 65% reduction in the 2-AAF induced-increase in the activities of these enzymes. Histological examination of the liver slices correlated with the changes in serum enzyme alterations. Similarly, kolaviron decreased the 2-AAF reduction of 5'-nucleotidase and glucose-6-phosphatase activities by 63% and 60%, respectively while BHA elicited 59% and 61% decrease in the activities of these enzymes. Simultaneous administration of kolaviron with 2-AAF inhibited microsomal lipid peroxidation as assessed by the thiobarbituric acid reacting substances (TBARS) formation by 66%. BHA produced a 64% reduction in TBARS formation. In the present study, kolaviron appears to act as an in vivo natural antioxidant and an effective hepatoprotective agent and is as effective as BHA.

Modulation of carbon tetrachloride-induced lipid peroxidation and xenobiotic-metabolizing enzymes in rats fed browned yam flour diet.

The modulatory effect of browned yam flour diet, a dietary, staple in south-western Nigeria, on carbon tetrachloride (CCl4)-mediated lipid peroxidation and on the activities of liver microsomal and cytosolic enzymes was studied in male rats. Browned yam flour diet fed at the level of 25% and 50% to rats for 5 weeks significantly reduced the lipid peroxidation induced by CCl4 (0.5 ml/kg/wk) administered two weeks after starting the animals with the diets. The diets elicited 62% and 79% reductions in CCl4-mediated peroxidation, respectively, in the absence of exogenously added oxidants. The activities of microsomal aniline hydroxylase (AH), aminopyrine-N-demethylase (APD), pentoxyresorufin-O-dealkylase (PROD) and cytosolic GSH S-transferase (GST) were increased when rats were fed the 25% or 50% browned yam flour diets. Browned yam flour fed at the level of 25% to rats decreased the CCl4-mediated reduction in the activities of microsomal AH, APD, PROD and GST by 64%, 28%, 58% and 25%, respectively, and by 82%, 48%, 83% and 55% when rats were fed with 50% of the diet. The results suggest that browned yam flour diet could protect against chemically-mediated lipid peroxidation and tissue damage possibly by scavenging chemically generated reactive species and enhancing carcinogen-detoxifying system.

Transcriptional and posttranscriptional regulation of insulin-like growth factor binding protein-3 by cyclic adenosine 3',5'-monophosphate: messenger RNA stabilization is accompanied by decreased binding of a 42-kDa protein to a uridine-rich domain in the 3'-untranslated region.

The Madin Darby bovine kidney (MDBK) cell line was used to investigate the mechanisms underlying the cAMP regulation of insulin-like growth factor binding protein-3 (IGFBP-3) gene expression. Treatment of confluent monolayers either with forskolin or cAMP produced a 60- to 75-fold induction of IGFBP-3 mRNA and protein levels. This effect did not require new protein synthesis as inhibition of translation by cycloheximide actually caused a 2-fold increase in the cAMP induction. The rates of IGFBP-3 gene transcription, assessed by nuclear run-on assays, increased approximately 15-fold in cells exposed to cAMP. In addition, the half-life of the IGFBP-3 mRNA transcript was increased approximately 3-fold in the presence of cAMP. Gel mobility shift and competition experiments revealed the specific binding of an approximately 42-kDa cytoplasmic protein factor to the 3'-untranslated region (3'-UTR) of the IGFBP-3 mRNA. A 21-nucleotide uridine-rich segment that contained no AUUUA motif was sufficient for the specific binding. The binding activity of this protein was reduced after cAMP treatment but was increased by phosphatase treatment. In conclusion, the cAMP induction of IGFBP-3 mRNA in MDBK cells occurred at both the transcriptional and posttranscriptional levels. The IGFBP-3 mRNA stabilization in MDBK cells probably involved the phosphorylation of a member of the family of U-rich region mRNA-binding proteins and is the first reported member whose RNA-binding activity is reduced by cAMP.

Possible modulatory effect of browned yam flour diet on chemically-induced toxicity in the rat.

The possible modulatory effect of browned yam flour, a local dietary staple in south western Nigeria, on the toxicity of 7,12-dimethylbenzanthracene (DMBA), 3-methylcholanthrene (3-MC), carbon tetrachloride (CCl4) and bromobenzene (BrB) in rats was investigated. Feeding rats with 25% browned yam flour 2 wk before treatment with 65 mg/kg DMBA (single dose) and 5 mg/kg 3-MC and continued for 3 wk significantly decreased the reduction in final body weight or weight gain and organ weights caused by the two compounds. Similarly, the diet decreased the reduction in body weight or weight gain and the increase in relative liver weight mediated by oral treatment with 0.5 ml CCl4/kg and 2.5 mmol BrB/kg body weight. Incorporation of 25% browned yam flour into rat diet significantly reduced the DMBA-mediated decrease in haemoglobin content, packed cell volume, red blood cell count and white blood cell count by 7, 5, 20 and 10%, respectively; while the diet reduced the 3-MC-mediated decrease in these parameters by 15, 28, 9 and 17%, respectively. The same diet elicited 23, 45, 13 and 33% decreases in CCl4 mediated reduction in these parameters and 23, 18, 16 and 29% in the case of BrB. Browned yam flour diet caused 10, 14 (P < 0.001) and 4% (P < 0.05) reductions in the DMBA-mediated increase in serum aspartate aminotransferase, alanine aminotransferase and serum alkaline phosphatase, respectively; and 32, 31 (P < 0.05) and 13% (P < 0.001) in the case of the 3-MC-mediated increase. Also, the diet reduced CCl4-mediated increase in the activities of these by 40, 34 and 31%, respectively and by 23, 30 and 29% following BrB treatment. These results suggest that browned yam flour diet could possibly be a modulator of chemically induced toxicity.

Induction of chromosomal aberrations, cytotoxicity, and morphological transformation in mammalian cells by the antiparasitic drug flubendazole and the antineoplastic drug harringtonine.

The antiparasitic drug flubendazole and the antineoplastic compound harringtonine were studied for ability to induce chromosomal damage in Chinese hamster lung (CHL) cells and cytotoxicity and morphological transformation in C3H/10T1/2 Cl 8 (10T1/2) mouse embryo fibroblasts. Flubendazole caused a dose- and time-dependent induction of polyploidy in CHL cells. In cells treated with 0.78 micrograms/ml flubendazole, the yield of polyploid cells was 95%. Harringtonine caused a dose- and time-dependent induction of chromosome breaks, and 0.195 micrograms/ml harringtonine induced chromosome breaks in 47% of CHL cells. Both flubendazole and harringtonine caused dose-dependent cytotoxicity to 10T1/2 cells at concentration ranges of 0.04-1.60 and 0.05-0.8 micrograms/ml, respectively. Flubendazole and harringtonine at concentrations of 0.08-0.4 and 0.4-0.8 micrograms/ml, respectively, induced morphological transformation (predominantly type II foci) in 10T1/2 cells. Three of four harringtonine-transformed cell lines and two of four flubendazole-transformed cell lines formed foci in reconstruction experiments with non-transformed 10T1/2 cells. All four harringtonine-transformed and all four flubendazole-transformed cell lines formed colonies in soft agar. Similar concentrations of flubendazole and harringtonine induced chromosome damage in CHL cells and cytotoxicity and morphological transformation in 10T1/2 cells. The ability of flubendazole to induce polyploidy may be part of the mechanism by which this compound induces morphological transformation. Similarly, the ability of harringtonine to induce chromosomal aberrations may be part of the mechanism by which this compound induces morphological transformation. Therefore, flubendazole and harringtonine induce cytotoxicity and morphological and anchorage-independent transformation, harringtonine induces chromosome aberrations (breakage, translocation, and rings), and flubendazole induces polyploidy in cultured mammalian cells. The clastogenic and cell transformation-inducing properties of these compounds suggest that these drugs may have carcinogenic potential. This should be investigated rigorously in animal carcinogenesis bioassays. The genotoxicity of these drugs should be considered during their development as antiparasitic and antineoplastic agents.

Mechanism of chemical carcinogenesis: an alternative hypothesis.

Ultimate carcinogens are usually very reactive electrophilic species and therefore of transient existence in the cell. Both because of the biological reactivity and the inherent danger in their reaction with DNA, direct interaction with the latter is prevented, especially in higher mammalian cells, by various cell defence mechanisms and, lastly, by a nuclear membrane monitoring system. Hence, reaction of chemical carcinogen with nuclear DNA is possible only when the cell is overwhelmed leading to cell death, or following a temporary breach of the nuclear membrane control points, but the DNA damage in the latter is totally reparable. Both situations are therefore of no consequence to chemical carcinogenesis. Instead, the excess electrical charge created by the physical presence of the electrophilic carcinogens is hypothesized to predispose the cell to irreversible genetic changes like cellular oncogene activation, or uncontrolled cell proliferation common to cancerous cells. These effects would be catalyzed by a Na+-H+ exchange-mediated intracellular alkalinization resulting from the cell's attempt to restore electrical neutrality by elevating the Na(+)-pump activity, fuelled by an increased Na+/H+ antiport.

In vitro O-demethylation from a heterocyclic nitrogen: a novel metabolic reaction?

1. O-Demethylation of 1-methoxyindole-3-carboxylic acid, in vitro, by determination of liberated formaldehyde, has been demonstrated using fortified 15,000 g rat liver supernatant fraction. 2. Results have been compared to those similarly obtained with a standard O-demethylation substrate, 4-nitroanisole, to substantiate and quantify the extent of the reaction. 3. Km values for 4-nitroanisole and methoxyindole-3-carboxylic acid were 8.9 and 6.5 mM, while Vmax values were 4.9, and 5.2 nmol HCHO produced/mg protein per min. 4. The significance of the metabolic O-demethylation reaction for N-methoxyindoles as a novel metabolic pathway is discussed in terms of pharmacological activity and biological reactivity.

Preparation of a flowable liquid bacterial insecticide based on Bacillus sphaericus.

Fermenter-produced Bacillus sphaericus 2362 was formulated into a thick, dark flowable liquid concentrate containing 4.8×10(9) c.f.u./ml and charcoal as protector against ultraviolet light. The potencies of the formulation against L4 Culex pipiens quinquefasciatus before and after storage for 2 years were 5714 and 5862 International Toxic Units (ITU), respectively, when compared with a standardized B. sphaericus from the WHO at 1000 ITU. In field trials, treatment at 1.01/ha gave 96 to 100% control of mosquito larvae. B. sphaericus could be re-isolated in 5% of the samples 9 months after application.

The metabolism of some 1-hydroxylated indoles in the rat.

A number of N-hydroxylated indole derivatives have been fed to rats and the urinary metabolites compared with those obtained on feeding the corresponding N-H indoles. 1-Methoxy- and 1-acetoxy-indole were metabolized to compounds normally observed on feeding indole. 1-Hydroxy- and 1-methoxy-indole-3-carboxylic acids were both partially excreted unchanged and reduced to indole-3-carboxylic acid which was excreted along with its glucuronide. Indole-3-glyoxylic acid and its 1-methoxy derivative were excreted almost entirely unchanged. 1-Hydroxyindole-3-glyoxylic acid was mostly excreted unchanged, but some reduction to indole-3-glyoxylic acid took place. The stability of these acids is associated with their low pKa values. The significance of the removal in vivo of groups attached through oxygen to the 1 position of indoles in relation to the synthesis of new potential medicinal agents is noted.