Protein tyrosine phosphatase 1B inhibitory activity of triterpenes isolated from Astilbe koreana

Bioassay-guided fractionation of a MeOH extract of the rhizomes of Astilbe koreana (Saxifragaceae), using an in vitro protein tyrosine phosphatase 1B (PTP1B) inhibitory assay, resulted in the isolation of a new triterpene, 3alpha,24-dihydroxyolean-12-en-27-oic acid (4), along with four triterpenes, 3-oxoolean-12-en-27-oic acid (1), 3beta-hydroxyolean-12-en-27-oic acid (beta-peltoboykinolic acid; 2), 3beta-hydroxyurs-12-en-27-oic acid (3), and 3beta,6beta-dihydroxyolean-12-en-27-oic acid (astilbic acid; 5). Compounds 1-5 inhibited PTP1B with IC50 values of 6.8+/-0.5, 5.2+/-0.5, 4.9+/-0.4, 11.7+/-0.9, and 12.8+/-1.1 microM, respectively. Our results indicate that 3-hydroxyl group and a carboxyl group in this type of triterpenes may be required for the activity, while addition of one more hydroxyl group at C-6 or C-24 may be responsible for a loss of activity. Thus, compounds 2 and 3 which possess only one hydroxyl group at C-3 and a carboxyl group at C-27 could be potential PTP1B inhibitors.     

Partial restoration of defective chloride conductance in DeltaF508 CF mice by trimethylamine oxide

This study was designed to test the in vivo efficacy of the chemical chaperone trimethylamine oxide (TMAO) in correcting the Cl- transport defect in a mouse model of cystic fibrosis (CF). Rectal potential difference (RPD) measurements were done in matched wild-type and DeltaF508 CF mice. Mice were treated by subcutaneous injections of TMAO. Wild-type mice demonstrated a forskolin-stimulated, Cl--dependent hyperpolarization of -6.4 +/- 0.8 mV (n = 11), which was significantly increased to -13.1 +/- 1.4 mV after treatment with TMAO. DeltaF508 CF mice showed no significant responses to forskolin. Treatment with TMAO recovered a forskolin-activated RPD in DeltaF508 CF mice (-1.1 +/- 0.2 mV; n = 17) but not in CFTR null mice. The effects of TMAO were dose dependent, resulting in a slope of -0.4 +/- 0.1 mV x g(-1) x kg(-1) in DeltaF508 CF mice. The forskolin-stimulated RPD in TMAO-treated DeltaF508 CF mice was partially blocked by glibenclamide and further stimulated by apigenin. The total response to forskolin plus apigenin was -2.5 +/- 0.45 mV (n = 6 mice), corresponding to 39% of the response evoked by forskolin only in wild-type mice.     

Estrogenic and cholinergic properties of the methanol extract of Ruellia praetermissa Sceinf. ex. Lindau (Acanthaceae) in female rats.

In search for alternative drugs with pharmacological profile to replace hormone replacement therapy, the effects of MeOH extract of Ruellia praetermissa on the uterus and gestation in rats was investigated. 350 mg/kg/day of extract from days 1-9, 1-17 and 9-17 respectively, resulted in increase of the number of implantation sites (56 to 64) and the percentage of implantation (68.6 +/- 2.7 to 90.5 +/- 0.5%). There was also an increase in body weight (1-9 and 1-17) followed by a slight decrease (154 +/- 15.5 to 125 +/- 10) in the body weight at term. The number and the mean value of corpora lutea per female decreased from 25.4 +/- 1.6 to 14.00 +/- 1.6. The extract produced dose-related contraction of the isolated uterine muscle in vitro comparable to ACh. Atropine in doses from 3.4 x 10(-6) to 3 x 10(-3) microM antagonized the response of the uterus to ACh at 2 microM. It induced an increase (0.03 +/- 0.002 to 0.34 +/- 0.001 g) of the uterine weight comparable to that produced by using 3 microM estradiol (0.03 +/- 0.001 to 0.35 +/- 0.005 g). It could therefore be postulated that this extract possesses estrogenic and possible cholinergic effects. The estrogenic effect could have been generated by plant sterols (beta-sitosterol and stigmasterol) and flavonoids (luteolin and apigenin) while cholinergic effect could be due to iridoid glycosides.     

Proinflammatory cytokine IL-1 beta promotes tumor growth of Lewis lung carcinoma by induction of angiogenic factors: in vivo analysis of tumor-stromal interaction

Inflammatory conditions are associated with tumor development. IL-1beta is a multifunctional and proinflammatory cytokine that affects nearly all types of cells. To investigate the role of IL-1beta in tumor growth in vivo, we transduced the retroviral vector coding human IL-1beta gene into mouse Lewis lung carcinoma (LLC) cells and subsequently inoculated the transformant (LLC/IL-1beta) to syngeneic C57BL/6 mice. Tumors derived from LLC/IL-1beta grew faster (240%, day 18, vs null-vector control LLC/neo; p < 0.01) and showed more abundant vasculature (250%, vs LLC/neo; p < 0.05), whereas LLC/IL-1beta cells, LLC/neo cells, and wild-type LLC cells did not show any significant difference in the growth rate in vitro. As compared with LLC/neo cells, LLC/IL-1beta cells secreted 2-fold the amount of vascular endothelial growth factor and >10-fold the amount of macrophage-inflammatory protein-2 (CXCL2), one of whose main functions is angiogenesis. Although LLC/IL-1beta itself did not secrete hepatocyte growth factor (HGF), the tumor derived from LLC/IL-1beta cells also contained a >4-fold higher concentration of HGF, another angiogenic factor. In situ hybridization of HGF mRNA in LLC/IL-1beta tumor sections demonstrated that stromal fibroblasts and infiltrating cells overexpressed HGF mRNA. Moreover, when cultured in the presence of HGF in vitro, LLC/IL-1beta cells secreted even larger amounts of vascular endothelial growth factor and macrophage-inflammatory protein-2. The antiangiogenic agent TNP-470 and anti-CXCR2 Ab inhibited the tumor growth of LLC/IL-1beta cells in vivo. These results indicated that secreting IL-1beta into the tumor milieu induces several angiogenic factors from tumor and stromal cells and thus promotes tumor growth through hyperneovascularization.     

Effects of exogenous thyroxine on C-2 and C-16 alpha hydroxylations of estradiol in humans

Thyroid dysfunction in humans is known to alter the excretory pattern of estrogen metabolites, suggesting that thyroid hormone directly influences the oxidative metabolism of estradiol. We examined the extent to which a brief period of hyperthyroidism specifically affected estradiol hydroxylation at C-2 and C-16 alpha, the two primary and competing sites of estrogen oxidation, using an in vivo radiometric assay in healthy male volunteers. Hydroxylation at C-2 was increased by a 2-week course of thyroxine (4.3 micrograms/kg/d) from 29.9% +/- 2.6% to 35.9% +/- 3.1% (P = 0.04), while 16 alpha-hydroxylation was unchanged (10.3% +/- 0.8% versus 9.3% +/- 0.5%). The greater extent of oxidation at C-2 was evidenced by a twofold increase in the urinary excretion of 2-hydroxyestrone (2.88 +/- 0.32 versus 5.30 +/- 0.85 micrograms/g creatinine), while the excreted products of 16 alpha-hydroxylation were unchanged. At the same time, significant reductions in total cholesterol (173.8 +/- 7.9 versus 139.4 +/- 8.9 mg/dl), low-density lipoprotein cholesterol (110.0 +/- 5.3 versus 83.8 +/- 7.7 mg/dl), and apolipoprotein B (68.2 +/- 3.3 versus 53.1 +/- 3.6 mg/dl) were observed. Serum levels of estrone, estradiol, sex hormone-binding globulin, high-density lipoprotein cholesterol, very low-density lipoprotein cholesterol, triglycerides, and apolipoprotein A-I were not significantly affected. This study adds to the growing evidence that catechol estrogen production in humans is more readily regulated than 16 alpha-hydroxylation, which is relatively refractory to treatment.     

Leuprolide acetate reduces both in vivo and in vitro ovarian steroidogenesis in infertile women undergoing assisted reproduction

Despite the probable inhibitory effects of GnRH analogues on ovarian steroidogenesis in vitro, their association with assisted reproduction protocols shows favorable results. This suggests that there are important differences in the behaviors of these drugs when administered in vivo versus in vitro. To clarify these differences, this study was designed to analyze the effect of leuprolide acetate (LA) on ovarian steroidogenesis in women undergoing In Vitro Fertilization (IVF). A prospective, randomized open label study was conducted on 14 women (26-35 years): seven receiving only gonadotrophins (Group 1) and seven receiving gonadotrophin plus LA at 1mg/day (Group 2). The LA in vivo effect was determined with serum and follicular fluid (FF) samples and via luteinized granulosa cell cultivation (GCC), where cells were obtained during oocyte retrieval after ovarian hyperstimulation. In vitro analysis was performed via addition of LA to GCC only for Group 1 (without LA) at progressively higher concentrations (0, 10(-12), 10(-9) and 10(-6)M). In vivo, the main observation was a reduction in androgen production in Group 2, represented by lower androstenedione production in FF (G1=6479+/-3458; G2=3021+/-1119 ng/ml; p=0.04) and a lower testosterone peak in GC at 96h (G1=0.64+/-0.12 ng/ml; G2=0.50+/-0.19 ng/ml; P=0.02), but a higher fertilization rate (G1=67%; G2=83%; p=0.009). In vitro, testosterone, estradiol and progesterone were also reduced by LA, even though this reduction occurred for progesterone only at the highest LA dosage (10(-6)M; 606.0+/-114.3 ng/ml versus 1524.0+/-246.5 ng/ml; p=0.02). Results show that LA reduces ovarian steroidogenesis in vivo by essentially inhibiting androgen synthesis; whereas, in vitro, ovarian steroidogenesis is reduced overall.     

Validation of a capillary electrophoresis method for the quantitative determination of free and total apigenin in extracts of Chamomilla recutita

A capillary electrophoretic method for the quantification of free and total apigenin in methanolic, ethanolic and glycolic extracts of Chamomilla recutita L. Rauschert (Asteraceae) is described. The method was validated for measurement of apigenin in the range 5.00-300 microg/mL (r2 = 0.993) and showed coefficients of intra-day (replicability) and inter-day (repeatability) variability of better than 2%. The limits of detection and quantification were 3.80 and 11.5 microg/mL, respectively, and the average recovery was 102.0 +/- 0.8% at three concentration levels of apigenin. Free and total apigenin contents in the extracts were, respectively, determined as 106 and 903 microg/g (methanolic extract), 77 and 817 microg/g (ethanolic extract) and 11.0 and 247 microg/g (glycolic extract).     

The effects of ACTH on adrenal steroidogenesis and blood corticosteroid levels in the echidna (Tachyglossus aculeatus).

1. The effects of short-term (S.T., 30 min) and long-term (L.T., 4 days) administration of ACTH on peripheral blood corticosteroid levels and on in vitro steroidogenesis were investigated. 2. Control levels of cortisol, corticosterone and aldosterone were 58 +/- 12, 130 +/- 26 and 10 +/- 6 (SEM) ng/100 ml respectively. 3. Corticosterone was 70% higher after S.T. and 150% higher after L.T., when cortisol was 800% higher. 4. Adrenal homogenates from control echidnas converted [14C]progesterone predominantly to 11-deoxycorticosterone (45%) and 11-deoxycortisol (12%). 5. After L.T. the principal product was corticosterone (25%), but S.T. had no effect. 6. In control echidnas the Km and V for 11 beta-hydroxylation of 11-deoxycorticosterone were 20 microM and 2.8 rho mol/min/mg respectively. After L.T. V increased to 10 rho mol/min/mg.     

Inhibition of rat testicular 17 alpha-hydroxylase and 17,20-lyase activities by anti-androgens (flutamide, hydroxyflutamide, RU23908, cyproterone acetate) in vitro

Flutamide, hydroxyflutamide, RU23908 and cyproterone acetate (CPA) inhibited rat testicular microsomal 17 alpha-hydroxylase and 17,20-lyase activities in vitro. The Km of [3H] progesterone for 17 alpha-hydroxylase was 45 +/- 0.62 nmol/l (+/- SEM, n = 12) and the Km of [3H] 17 alpha-hydroxyprogesterone for 17,20-lyase was 192 +/- 0.42 nmol/l (+/- SEM, n = 12). The Ki values for 17 alpha-hydroxylase, determined from Lineweaver-Burk plots were 102 +/- 3.2 mumol/l (+/- SEM, n = 6), 363 +/- 3.8 mumol/l (+/- SEM, n = 6), 118 +/- 1.4 mumol/l (+/- SEM, n = 6) and 123 +/- 2.1 mumol/l (+/- SEM, n = 6) for flutamide, hydroxyflutamide, RU23908 and CPA respectively. Flutamide and CPA were mixed-type inhibitors, whereas hydroxyflutamide and RU23908 were competitive inhibitors of 17 alpha-hydroxylase activity. Ki values for 17,20-lyase were 33 +/- 3.1 mumol/l (+/- SEM, n = 6), 112 +/- 3.1 mumol/l (+/- SEM, n = 6), 69 +/- 4.4 mumol/l (+/- SEM, n = 6) and 71 +/- 3.2 mumol/l (+/- SEM, n = 6) for flutamide, hydroxyflutamide, RU23908 and CPA, respectively. Inhibition was found to be competitive in each case. Although the characteristic action of anti-androgens is at the receptor level, these results demonstrate that anti-androgens may also have inhibitory effects on androgen biosynthesis which could prove to be of clinical significance.     

Carbon isotope effects on the pyruvate dehydrogenase reaction and their importance for relative carbon-13 depletion in lipids

A method has been developed for the positional 13C isotope analysis of pyruvate and acetate by stepwise quantitative degradation. On its base, the kinetic isotope effects on the pyruvate dehydrogenase reaction (enzymes from Escherichia coli and Saccharomyces cerevisiae) for both of the carbon atoms involved in the bond scission (double isotope effect determination) and on C-3 of pyruvate have been determined. The experimental k12/k13 values with the enzyme from E. coli on C-1 and C-2 of pyruvate are 1.0093 +/- 0.0007 and 1.0213 +/- 0.0017, respectively, and, with the enzyme from S. cerevisiae, the values are 1.0238 +/- 0.0013 and 1.0254 +/- 0.0016, respectively. A secondary isotope effect of 1.0031 +/- 0.0009 on C-3 (CH3-group) was found with both enzymes. The size of the isotope on C-1 indicates that decarboxylation is more rate-determining with the yeast enzyme than with the enzyme from E. coli, although it is not the entirely rate-limiting step in the overall reaction sequence. Assuming appropriate values for the intrinsic isotope effect on the decarboxylation step (k3) and the equilibrium isotope effect on the reversible substrate binding (k1, k2), one can calculate values for the partitioning factor R (k3/k2: E. coli enzyme 4.67, S. cerevisiae enzyme 1.14) and the intrinsic isotope effects related to the carbonyl-C (k1/k'1 = 1.019; k3/k'3 = 1.033). The isotope fractionation at C-2 of pyruvate gives strong evidence that the well known relative carbon-13 depletion in lipids from biological material is mainly caused by the isotope effect on the pyruvate dehydrogenase reaction. In addition, our results indicate an alternating 13C abundance in fatty acids, that has already been verified in some cases.     

Hormone secretion by preimplantation embryos in a dynamic in vitro culture system.

Bovine embryos recovered from superovulated donors on Days 8-18 postestrus were cultured in vitro in a tissue perifusion system to quantify hormone secretion. Embryos were cultured for 24 h at 37 degrees C in Ham's F-10 medium supplemented 5% v/v with heat-treated, charcoal-stripped calf serum; 100 IU/ml penicillin; and 100 micrograms/ml streptomycin. The medium was saturated with 5% CO2 in air and perifused at 50 microliters/min (3 ml/h). Estrone (E1) estradiol (E2), progesterone (P4), prostaglandin E2 (PGE2), and prostacyclin (PGI2) were quantified by RIA in 6-h pools of perifusate fractions. Estrone was measurable (pg/h/embryo; mean +/- SE) on Days 13 (10.80 +/- 4.56) and 15 (34.80 +/- 9.80); E2 on Days 11 (36.80), 12 (81.28 +/- 29.80), 13 (11.75 +/- 4.09), 15 (157.20 +/- 112.60), and 16 (30.26 +/- 8.76); and P4 (ng/h/embryo) on Days 13 (0.5-1.0) and 17 (approximately 1.5). PGE2 was secreted by Day 10 bovine embryos during the last 6 h of culture (19-24 h) and throughout culture for Day 11-18 embryos. The rate of PGE2 secretion increased (p less than 0.05) over the previous days(s) at Days 13 and 17. The mean (+/- SE) secretion rates (pg/h/embryo) for the 24-h culture by embryonic ages were as follows: Day 11 (63.39 +/- 14.61), 12 (172.10 +/- 30.90), 13 (3094.08 +/- 283.35), 14 (1633.89 +/- 49.98), 15 (3739.23 +/- 1082.79), 16 (4955.37 +/- 1381.83), 17 (11893.23 +/- 1188.48), and 18 (13827.99 +/- 3587.88).(ABSTRACT TRUNCATED AT 250 WORDS)     

5 alpha-androstane-3 beta,17 beta-diol hydroxylating enzymes in stroma and epithelium of human benign prostatic hyperplasia (BPH)

As enzymatic hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol) may be a factor in controlling the 5 alpha-dihydrotestosterone (DHT) content in the prostate, we were interested in activity and distribution of these enzymes in epithelium and stroma of human benign prostatic hyperplasia (BPH). The enzyme activities were measured after mechanical separation of BPH tissue from 15 patients of various ages into stroma and epithelium, and optimization of the in vitro transformation of 3 beta-diol to hydroxylated products, which were analyzed by HPLC. The main results were: (1) 3 beta-diol was hydroxylated at C-7 alpha, C-7 beta, C-6 alpha, and C-6 beta. (2) The mean Michaelis constant Km (nM +/- SEM) for hydroxylation at C-7 alpha(beta) (168 +/- 21) was significantly lower than at C-6 alpha(beta) (601 +/- 43) without differences between stroma and epithelium. (3) Hydroxylation at alpha position dominated significantly over that at beta. (4) The mean maximal metabolic rate Vmax (pmol . mg protein-1 . h-1) of hydroxylation at C-6 alpha was about 7-fold lower in stroma (3.4 +/- 0.2) than in epithelium (23.8 +/- 4.1), concerning the other hydroxylations, Vmax was about 1.6-fold lower in stroma. (5) With increasing age of the patients there was a significant decrease of the 3 beta-diol hydroxylation in stroma and epithelium. It is discussed that the significantly lower activity of 3 beta-diol hydroxylation in stroma compared to epithelium and the decrease of activity with increasing age might potentiate the DHT accumulation in stroma of BPH.     

Alterations in the intermediary metabolism of selenium-deficient mice

Male albino mice were pair-fed a torula yeast-based selenium-deficient (Se-) diet containing 10 ppb selenium for 4 months, while a control group (Se+) received a similar diet supplemented with 330 ppb selenium as Na2SeO3. In addition to previously observed modulations of drug-metabolizing enzymes (Reiter, R. and Wendel, A. (1985) Biochem. Pharmacol. 34, 2287-2290), an increase of 6-phosphogluconate dehydrogenase activity and succinate dehydrogenase activity in liver by about 60% was found. In vivo, an increased 14CO2 exhalation from a tracer dose of glucose either labeled in the C-1- or C-6 position was observed in selenium-deficient mice. However, no difference in the total CO2 exhalation of Se(-)- as compared to Se+-mice was detectable. In line with the assumption that Se(-)-mice have an increased glucose turnover, Se(-)-mice exhibited a greater glucose tolerance when treated with an oral glucose load of 2.5 mg glucose/kg body weight. Also, the Se(-)-mice had a lower blood glucose level as compared to Se+-controls (89 +/- 3 versus 110 +/- 12 mg glucose/100 ml blood). Further in vitro experiments with red blood cells from Se(-)-mice showed that erythrocytes did not contribute to an increased CO2 formation from glucose via the pentose phosphate shunt. No significant differences between Se(-)- and Se+-animals were found in the profile of urinary metabolites, including ketone bodies and nitrogen excretion. These findings suggest a hitherto unknown involvement of selenium in specific regulatory sites of intermediary metabolism.     

Identification of the novel splicing variants for the hPXR in human livers

Chronic anthracycline administration to rabbits causes impairment of cardiac contractility and decreased gene expression of the calcium-induced calcium release channel of sarcoplasmic reticulum (SR), the ryanodine receptor (RYR2). The C-13 hydroxy metabolite (doxorubicinol), formed in the heart, has been hypothesized to contribute to anthracycline cardiotoxicity. C-13 deoxydoxorubicin is an analog unable to form the C-13 hydroxy metabolite. Therefore, doxorubicin, C-13 deoxydoxorubicin, or saline was administered to rabbits (1 mg/kg iv twice weekly for 8 weeks). Left ventricular fractional shortening (LVFS) was decreased by chronic treatment with doxorubicin (28 +/- 2%; P < 0.05), but not C-13 deoxydoxorubicin (33 +/- 2%) compared to age-matched pair-fed controls. Doxorubicin, but not C-13 deoxydoxorubicin, caused a significant reduction (P < 0.02) in the ratio of RYR2/Ca-Mg ATPase (SERCA2) mRNA levels (0.57 +/- 0.1 vs 1.22 +/- 0.2, respectively) in the left ventricle. This suggests that doxorubicinol may contribute to the downregulation of cardiac RYR2 expression in chronic doxorubicin cardiotoxicity.     

New steroidal glycosides from rhizomes of Clintonia udensis

A total of 10 steroidal glycosides, together with three new spirostanol glycosides (6-8), a new furostanol glycoside (9), and a new cholestane glycoside (10), were isolated from the rhizomes of Clintonia udensis (Liliaceae). The structures of the new compounds were determined on the basis of extensive spectroscopic analyses, including 2-D nuclear magnetic resonance (NMR) data, and of hydrolytic cleavage followed by chromatographic or spectroscopic analyses. The isolated glycosides were evaluated for their cytotoxic activity against HL-60 leukemia cells. Spirostanol glycosides 1 and 2, and furostanol glycoside 4 showed cytotoxic activity with IC(50) values of 3.2+/-0.02, 2.2+/-0.12, and 2.2+/-0.06 microg/ml, respectively. Neither the spirostanol and furostanol saponins with a hydroxy group at C-1 (6 and 9) and C-12 (7 and 8) nor cholestane glycosides (5 and 10) exhibited apparent cytotoxic activity at a sample concentration of 10 microg/ml.     

Molecular cloning, expression and characterization of a glycosyltransferase from rice

Secondary plant metabolites undergo several modification reactions, including glycosylation. Glycosylation, which is mediated by UDP-glycosyltransferase (UGT), plays a role in the storage of secondary metabolites and in defending plants against stress. In this study, we cloned one of the glycosyltransferases from rice, RUGT-5 resulting in 40–42% sequence homology with UGTs from other plants. RUGT-5 was functionally expressed as a glutathione S-transferase fusion protein in Escherichia coli and was then purified. Eight different flavonoids were used as tentative substrates. HPLC profiling of reaction products displayed at least two peaks. Glycosylation positions were located at the hydroxyl groups at C-3, C-7 or C-4′ flavonoid positions. The most efficient substrate was kaempferol, followed by apigenin, genistein and luteolin, in that order. According to in vitro results and the composition of rice flavonoids the in vivo substrate of RUGT-5 was predicted to be kaempferol or apigenin. To our knowledge, this is the first time that the function of a rice UGT has been characterized.     

Regio and stereospecific DNA adduct formation in mouse lung at N6 and N7 position of adenine and guanine after 1,3 butadiene inhalation exposure

Butadiene monoepoxide (BMO) alkylated guanine N7 and adenine N 6 adducts were prepared and enriched by solid phase extraction and HPLC. The purified adducts were analysed by a modified 32P-postlabelling assay, which utilized one dimensional TLC chromatography and a subsequent HPLC analysis with UV and radioactivity detectors. In vitro with Ct-DNA the formation of N7-dGMP and N 6-dAMP adducts were linear at a concentration range of 44 to 870 nmol of BMO per mg DNA at physiological pH. N7- dGMP and N 6-dAMP adducts were formed in a ratio of 200:1. In dGMP and in dAMP 48 % and 86 % of adducts were covalently bound to the C-2 carbon of BMO. CD-1 mice were inhalation exposed to butadiene for 5 days and 6 h per day. The N7-dGMP adduct level in lung samples of animals exposed to 200, 500 and 1300 ppm was 2.8 +/- 0.9 fmol, 11 +/- 2.0 fmol and 30 +/- 6.7 fmol in 10 mug DNA, respectively. The level of N 6-dAMP adducts in lung samples after 500 ppm and 1300 ppm exposure was 0.09 +/- 0.06 fmol and 0.11 +/- 0.05 fmol in 10 mug DNA. At 200 ppm the adduct level was below the detection limit. A sub-group of animals exposed to 1300 ppm was killed 3 weeks after the last exposure. N7-dGMP adducts were not detected but the level of N 6-dAMP adducts was not affected. N7-dGMP adducts were formed in a clear stereospecific manner in vivo. S -BMO adducts were the main product and represented 77 % (n = 4, SD = 2%) of total BMO adducts. No clear conclusion can be drawn about the enantiospecific DNA binding at the N 6 position of dAMP, because of the poor separation of the enantiomers. However, we could separate regioisomeric adducts which indicated that C-2 adducts represented 69 +/- 3 % of the total N 6 adducts formed in mice lung DNA. This observation is supported by the data derived from in vitro DNA experiments but is different to our previously published data, which indicates the 2:1 (C-1:C-2) ratio in regioisomer formation in nucleotides or nucleosides. We suggest that the data presented in this communication indicate a different mechanism between nucleotides and DNA in BMO-derived adduct formation- Dimroth rearrangement dominates in nucleotides, but in double stranded DNA a direct alkylation is probably the major mechanism of adduct formation.     

Regio and stereospecific DNA adduct formation in mouse lung at N6 and N7 position of adenine and guanine after 1,3 butadiene inhalation exposure

Butadiene monoepoxide (BMO) alkylated guanine N7 and adenine N 6 adducts were prepared and enriched by solid phase extraction and HPLC. The purified adducts were analysed by a modified 32P-postlabelling assay, which utilized one dimensional TLC chromatography and a subsequent HPLC analysis with UV and radioactivity detectors. In vitro with Ct-DNA the formation of N7-dGMP and N 6-dAMP adducts were linear at a concentration range of 44 to 870 nmol of BMO per mg DNA at physiological pH. N7- dGMP and N 6-dAMP adducts were formed in a ratio of 200:1. In dGMP and in dAMP 48 % and 86 % of adducts were covalently bound to the C-2 carbon of BMO. CD-1 mice were inhalation exposed to butadiene for 5 days and 6 h per day. The N7-dGMP adduct level in lung samples of animals exposed to 200, 500 and 1300 ppm was 2.8 +/- 0.9 fmol, 11 +/- 2.0 fmol and 30 +/- 6.7 fmol in 10 mug DNA, respectively. The level of N 6-dAMP adducts in lung samples after 500 ppm and 1300 ppm exposure was 0.09 +/- 0.06 fmol and 0.11 +/- 0.05 fmol in 10 mug DNA. At 200 ppm the adduct level was below the detection limit. A sub-group of animals exposed to 1300 ppm was killed 3 weeks after the last exposure. N7-dGMP adducts were not detected but the level of N 6-dAMP adducts was not affected. N7-dGMP adducts were formed in a clear stereospecific manner in vivo . S -BMO adducts were the main product and represented 77 % ( n = 4, SD = 2%) of total BMO adducts. No clear conclusion can be drawn about the enantiospecific DNA binding at the N 6 position of dAMP, because of the poor separation of the enantiomers. However, we could separate regioisomeric adducts which indicated that C-2 adducts represented 69 +/- 3 % of the total N 6 adducts formed in mice lung DNA. This observation is supported by the data derived from in vitro DNA experiments but is different to our previously published data, which indicates the 2:1 (C-1:C-2) ratio in regioisomer formation in nucleotides or nucleosides. We suggest that the data presented in this communication indicate a different mechanism between nucleotides and DNA in BMO-derived adduct formation- Dimroth rearrangement dominates in nucleotides, but in double stranded DNA a direct alkylation is probably the major mechanism of adduct formation.     

Pathways of glycogen synthesis from glucose during the glycogenic response to insulin in cultured foetal hepatocytes.

The pathways of glycogen synthesis from glucose were studied using double-isotope procedures in 18-day cultured foetal-rat hepatocytes in which glycogenesis is strongly stimulated by insulin. When the medium containing 4 mM-glucose was supplemented with [2-3H,U-14C]glucose or [3-3H,U-14C]glucose, the ratios of 3H/14C in glycogen relative to that in glucose were 0.23 +/- 0.04 (n = 6) and 0.63 +/- 0.09 (n = 8) respectively after 2 h. This indicates that more than 75% of glucose was first metabolized to fructose 6-phosphate, whereas 40% reached the step of the triose phosphates prior to incorporation into glycogen. The stimulatory effect of 10 nM-insulin on glycogenesis (4-fold) was accompanied by a significant increase in the (3H/14C in glycogen)/(3H/14C in glucose) ratio with 3H in the C-2 position (0.29 +/- 0.05, n = 6, P less than 0.001) or in the C-3 position (0.68 +/- 0.09, n = 8, P less than 0.01) of glucose, whereas the effect of a 12 mM-glucose load (3.5-fold) did not alter these ratios. Fructose (4 mM) displaced [U-14C]glucose during labelling of glycogen in the presence and absence of insulin by 50 and 20% respectively, and produced under both conditions a similar increase (45%) in the (3H/14C in glycogen)/(3H/14C in glucose) ratio when 3H was in the C-2 position. 3-Mercaptopicolinate (1 mM), an inhibitor of gluconeogenesis from lactate/pyruvate, further decreased the already poor labelling of glycogen from [U-14C]alanine, whereas it increased both glycogen content and incorporation of label from [U-14C]serine and [U-14C]glucose with no effect on the relative 3H/14C ratios in glycogen and glucose with 3H in the C-3 position of glucose. These results indicate that an alternative pathway in addition to direct glucose incorporation is involved in glycogen synthesis in cultured foetal hepatocytes, but that insulin preferentially favours the classical direct route. The alternative foetal pathway does not require gluconeogenesis from pyruvate-derived metabolites, contrary to the situation in the adult liver.