Biomedical subjects
N Bjelogrlic
Publications and source records attributed to N Bjelogrlic.
TPA decreases the p53 response to benzo[a]pyrene-DNA adducts in vivo in mouse skin.
TPA, a well-known tumor promoter, decreased the response of nuclear p53 immunoreactivity to benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)-DNA adducts in C57BL/6 mouse skin in vivo. A dose-dependent increase in both the level of BPDE-DNA adducts and nuclear p53 immunoreactivity was found in mice treated topically with 50-750 microg benzo[a]pyrene. Such a positive correlation between the adducts and p53 positivity was suggested by an earlier study. Since p53 probably functions in DNA damage control, interference by TPA with the p53 response could be a mechanism in TPA-induced tumor promotion. Whether such a mechanism is more general in tumor promotion deserves further study.
Involvement of P450 1A1 in benzo(a)pyrene but not in benzo(a)pyrene-7,8-dihydrodiol activation by 3-methylcholanthrene-induced mouse liver microsomes.
Synchronous fluorescence spectrophotometry for benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE)-DNA adducts was used to study the activation pathway of benzo(a)pyrene in C57BL/6 mice. Benzo(a)pyrene but not benzo(a)pyrene-7,8-diol activation by 3-methylcholanthrene-induced mouse liver microsomes was inhibited by a monoclonal antibody (Mab 1-7-1) against CYP1A1/2 suggesting that 1A1 probably takes part in the first P450 reaction. However, aryl hydrocarbon hydroxylase activity, a classical measure of benzo(a)pyrene metabolism, was not inhibited by the same concentration of Mab 1-7-1. None of the other antibodies used, detecting 2A, 2B, 2C or 2E subfamilies, inhibited the adduct formation. Troleandomycin and gestodene, chemical inhibitors of human 3A4, inhibited benzo(a)pyrene-7,8-diol activation by 3-methylcholanthrene-induced microsomes to some extent only in high concentrations. Although liver microsomes from 3-methylcholanthrene-induced mice catalyzed the formation of BPDE-DNA in vitro clearly more than uninduced microsomes, 3-methylcholanthrene pretreatment in vivo decreased the adduct formation in benzo(a)pyrene-treated mice. These results emphasize the significance of detoxicating and DNA-repairing pathways in vivo. Finally, synchronous fluorescence spectrophotometry for BPDE-DNA measures the end-point of the three-step activation pathway while aryl hydrocarbon hydroxylase measures a one-step hydroxylation. Thus, these methods should be used rather as corroborative than mutually exclusive assays.
Benzo[a]pyrene-globin adducts detected by synchronous fluorescence spectrophotometry: method development and relation to lung DNA adducts in mice.
A simple synchronous fluorescence spectrophotometry (SFS) to detect benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)-globin adducts is described. SFS for BPDE-DNA, which measures detached benzo[a]pyrene (B[a]P)-tetrols after acid hydrolysis of DNA, was applied for BPDE-globin adducts in B[a]P-treated C57BL/6 (B6) mice. Unlike DNA samples, globin is not measurable as such after acid hydrolysis because proteins give a background in SFS. Furthermore, proteinase incubation before acid hydrolysis of globin gave too much background even after purification to be useful in this assay. Of several purification procedures tried after acid hydrolysis (protein precipitation, elution through Sep-Pak C18, filtration, ether extraction of tetrols), the lowest background fluorescence was obtained with ether extractions of B[a] moieties. Ether phases were evaporated to dryness and the remainder dissolved in distilled water (1 ml), which was measured by SFS. Compared to DNA, somewhat milder hydrolysis conditions were optimal for globin samples (0.05 M HCl, 1.5 h, + 90 degrees C). Globin samples from B[a]P-treated mice gave a peak at the same wavelength (345 nm excitation) as the hydrolysis products of BPDE-DNA adducts, indicating B[a]P-tetrols and triols in the sample. Less than half of B[a]P measured in globin was from covalently bound BPDE. In mice injected i.p. with 1-160 mg/kg of B[a]P there was a dose-dependent increase in the amount of BPDE adducts in globin and a positive correlation with lung and liver DNA. Globin adducts were a more sensitive indicator of B[a]P exposure than DNA adducts because more globin can be used for the assay. Although both covalently and non-covalently bound BPDE in globin are detected by SFS, this method is the simplest described so far, reproducible and theoretically sensitive enough for human biomonitoring.
Diurnal variation in the concentrations of catecholamines and indoleamines in the median eminence and in the intermediate and posterior lobes of the pituitary gland of the male rat.
Diurnal changes in monoamine concentrations were studied in the median eminence and in the intermediate and posterior lobes of the pituitary gland of the male rat. The concentrations of serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), noradrenaline (NA) and dopamine (DA) were analyzed at 7 time points over a 24-h period. Diurnal variation was analyzed by analysis of variance (ANOVA) with time of day as a class variable as well as by 24 h and 12 h cosine curve fittings. There were marked time-dependent changes in the median eminence concentrations of 5-HT (ANOVA: P = 0.0085), 5-HIAA (P = 0.003) and NA (P = 0.0003). Cosine curves with 24-h periods fit the data points with peaks around 13.00 h. DA levels also varied with an apparent 24-h rhythm in the median eminence, but the changes did not reach the level of significance in the ANOVA. In the intermediate lobe of the pituitary gland, the concentrations of DA varied significantly during the course of the 24-h cycle (P = 0.0011) and were well-fitted to a 24-h cosine wave. The 5-HIAA levels also showed marked diurnal changes (P = 0.025) with an evident 12-h rhythm. In contrast, NA and 5-HT concentrations did not appear to vary during the 24-h cycle. In the posterior lobe of the pituitary gland. DA had a 24-h rhythm (P = 0.0005) similar to the intermediate lobe. NA and indoleamine levels did not show any significant variation.(ABSTRACT TRUNCATED AT 250 WORDS)
Benzo[a]pyrene-DNA-adducts and monooxygenase activities in mice treated with benzo[a]pyrene, cigarette smoke or cigarette smoke condensate.
Synchronous fluorescence spectrophotometry (SFS), developed to study benzo[a]pyrene-7,8-diol-9,10-epoxide(BPDE)-DNA, was used to measure the in vivo formation of DNA-adducts in genetically responsive C57BL/6 (B6) and non-responsive DBA/2 (D2) mice. Treatment with cigarette smoke by inhalation for 3-16 days, or i.p. injection of cigarette smoke condensate or neutral fraction did not lead to detectable levels of BPDE-DNA-adducts in either lungs or liver, although aryl hydrocarbon hydroxylase (AHH) activity, an indicator of benzo[a]pyrene (BP) metabolism, was clearly induced in lungs of B6 mouse. A dose-dependent amount of BPDE-DNA-adducts in lung and somewhat less in liver was found after i.p. injection with BP (20-80 mg/kg). Mice treated with vehicle or 4 mg/kg of BP were negative for adducts by SFS. In B6 mice AHH was induced both in lungs and livers while there was no AHH induction in D2 mice although the levels of BPDE-DNA-adducts were somewhat higher than in B6 mice. Thus, no clear correlation seems to exist between AHH activity and the formation of BPDE-DNA-adducts. Also, according to our results SFS can be used to quantitate adduct-formation in in vivo animal studies.
Serotonin turnover in discrete hypothalamic nuclei and mesencephalic raphe nuclei of young and adult spontaneously hypertensive rats.
Serotonin levels and turnover were analyzed in discrete forebrain and mesencephalic nuclei of young (4-week-old) and adult (14-week-old) spontaneously hypertensive rats and age-matched normotensive control Wistar Kyoto rats. Most changes observed were age-dependent, and occurred only in young, early hypertensive rats. Both serotonin levels and the accumulation rate of 5-hydroxy-tryptophan after L-amino acid decarboxylase inhibition were higher in the nuclei periventricularis and paraventricularis of the hypothalamus of young hypertensive rats than in controls. In addition, 4-week-old spontaneously hypertensive rats showed higher 5-hydroxytryptophan accumulation rates in the nuclei supraopticus and dorsomedialis of the hypothalamus than controls. The only difference in serotonin metabolism found in adult hypertensive rats was high serotonin concentration in the median eminence of the hypertensive animals. Our results suggest the presence of anatomically specific, age-dependent alterations in serotonin metabolism, localized to selected hypothalamic nuclei in young hypertensive rats. These data support a role for the hypothalamic serotonin in the development of the spontaneous (genetic) hypertension in the rat.