Biomedical subjects
Publications and source records attributed to N Nemoto.
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Modulation of microsome mediated benzo[a]pyrene (BP) metabolism by oleic and linoleic acids was studied. Oleic and linoleic acids did not influence the apparent activity of aryl hydrocarbon hydroxylase or the relative ratios of BP-7,8-dihydrodiol to phenol metabolites on high performance liquid chromatography. The total binding of BP-metabolites to DNA was not influenced much by the presence of oleic acid, but was inhibited significantly by linoleic acid (p less than 0.05). The quantity of BP-metabolite bound adducts was determined by Sephadex LH-20 column chromatography with a methanol gradient. The amount of adducts bound with the anti-isomer of BP-7,8-dihydrodiol-9,10-oxide (diol-epoxide) was more than that with 9-hydroxybenzo[a]pyrene-4,5-oxide in the absence of fatty acids, and vice versa in the presence of fatty acids. The presence of 1, 2 or 5 microliters/ml oleic acid decreased the amounts of adducts with the anti- and syn-isomers of the diol-epoxide but not those with 9-hydroxybenzo[a]pyrene-4,5-oxides, whereas the presence of 0.08 - 2 microliters/ml of linoleic acid decreased the amounts of all these adducts but to different extents, resulting in predominance of 9-hydroxybenzo[a]pyrene-4,5-oxide derived adducts over anti-isomer adducts of the diol-epoxide. These observations suggest that endogenous materials that do not have any enzyme activity may have important influences on the metabolism of chemical carcinogens.
The effects of Brij 58 on microsome-mediated benzo[a]pyrene (BP)-metabolism were investigated. At a concentration of 0.01%, which did not decompose the microsomal mixed-function oxidase system, Brij 58 increased UDP-glucuronyl transferase activity on 3-hydroxy-BP to 560% of the control level. Quantitative changes of BP-metabolites by glucuronidation were found by high performance liquid chromatography. On incubation in the presence of UDP-glucuronic acid, the amounts of phenols decreased but the amounts of dihydrodiols did not change. Brij 58 enhanced the decrease in phenol metabolites. Unexpectedly, the binding of BP-metabolites to DNA was found to be higher in the presence of UDP-glucuronic acid, and Brij 58 also enhanced the binding in the presence of UDP-glucuronic acid. The ratio of bound adducts with BP-7,8-dihydrodiol-9,10-oxide to those with 9-hydroxy-BP-4,5-oxide, which were separated by Sephadex LH-20 column chromatography, was increased by UDP-glucuronic acid and further enhanced by Brij 58. These results are discussed in relation to the role of glucuronidation in metabolism of BP.
Detection of T4 and T3 in paraffin sections of the rat thyroid gland in various functional states was attempted by an immunoenzyme technique. Both hormones showed a similar localization, being detected mainly in the colloid in the follicular lumen, and sometimes in certain regions of the cytoplasm of the follicular epithelium. Following administration of TSH, their stainability was increased, and the localization within the cytoplasm became more remarkable. Decrease or disappearance of stainability was observed after hypophysectomy or administration of PTU. These findings support the view that the immunostainability of T4 and T3 is closely correlated with the functional state of the thyroid gland.
Genetic differences in metabolism of benzo(a)pyrene (BP) were investigated using cultured epidermal keratinocytes and dermal fibroblasts isolated from four inbred strains of mice: C3H/HeJms and C57BL/6J that are responsive; and DBA/2 and AKR/Jms that are nonresponsive in terms of inducibility of aryl hydrocarbon hydroxylase (AHH) activity. Primary cultures of epidermal and dermal cells isolated from newborn mice were treated with benz(a)anthracene for 24 hr. In both types of cells, AHH activity was induced in all four strains of mice, irrespective of their responsiveness in vivo. In the epidermal cells, basal AHH activity varied within a relatively small range of 4.6 to 8.8 pmol per mg protein per hr, and the activity was increased 4.4 to 8.7 times by benz(a)anthracene treatment. There was no difference in the extent of the induction in different strains of mice. In dermal cells, the basal level of AHH activity was in the range of 3.6 to 9.1 pmol per mg protein per hr, and benz(a)anthracene treatment induced AHH, but to various degrees depending on the responsiveness in vivo. Epidermal cells of the responsive mice metabolized more than 90% of the added BP in 48 hr, while those of nonresponsive mice metabolized only 60 to 70%. Dermal cells also metabolized BP, but to a lesser extent than did epidermal cells, and there was no strain difference in its metabolism. Time-course studies revealed that epidermal cells of responsive mice metabolized BP more rapidly than did those of nonresponsive mice. The activities of epidermal and dermal cells to metabolize BP were further demonstrated by a cell-mediated mutation assay. Consistent with the results of time-course analysis of the metabolites, shorter treatments with BP (less than 24 hr) showed a clear difference in BP metabolism associated with responsiveness.
The metabolism of chemical carcinogens by a human hepatoma cell line, huH-1, was studied. The huH-1 line has been derived from a hepatoma of a 57-year-old HBs-antigen carrier and cultivated for several years. The hepatoma cells metabolized about 90% of 5 microM benzo[a]pyrene into water-soluble products within 24 h. Aryl hydrocarbon hydroxylase activity in huH-1 cells was induced to 24 times higher than the basal level by treatment with 13 microM benz[a]anthracene for 24 h. Metabolic activation of benzo[a]pyrene, dimethylnitrosamine and aflatoxin B1 by huH-1 cells was observed by cell-mediated sister-chromatid exchange assay. Sister-chromatid exchanges in human diploid fibroblasts were observed in the cultures mixed with or without huH-1 cells. All 3 chemicals induced sister-chromatid exchanges in human fibroblasts far more efficiently in the cultures mixed with huH-1 cells than in those without huH-1 cells. Some characteristics of huH-1 cells as a human cell-mediated metabolic activation system for carcinogens are discussed.
The influence of 3-methyl-4-(dimethylamino)azobenzene(3'-Me-DAB) on the drug-metabolizing system in the liver was investigated. Feeding of 3-Me-DAB for 3 weeks at 10, 20 and 600 ppm increased the content of hepatic microsomal cytochrome P-450 slightly (up to 27% raise) but significantly. The feeding effects were also demonstrated in S-9 activity (up to 91% raise) when the mutagenicity of 3-methylcholanthrene (3-MC) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) was assayed in Ames system using S-9 fraction from 3'-Me-DAB-treated rat livers.
The kinetics of glucuronidation with 3- and 9-hydroxybenzo(a)pyrene (3-OH BP and 9-OH BP) were studied using rat and mouse liver microsomes. The UDP-glucuronyl transferase had two apparent Km values for 3- and 9-OH BP in both species, suggesting that the hepatic enzyme is efficient in glucuronidation of first-phase metabolites, especially at low concentrations of the carcinogen.
The effects of serum on the metabolism of benzo[a]pyrene (BP) by liver microsomes from 3-methylcholanthrene-treated rats were studied. In the presence of serum, the aryl hydrocarbon hydroxylase activities were enhanced to 160-570% of the control value. Enhancement of BP-metabolism by serum was also revealed by high performance liquid chromatography, but the relative amounts of a series of BP-metabolites were not changed appreciably by addition of serum. Binding of BP-metabolites to DNA was influenced by serum: adducts derived from two stereo-isomeric BP-7,8-di-hydrodiol-9,10-oxides were increased, while those from 9-hydroxybenzo[a]pyrene-4,5-oxide were reduced.
The occurrence of alpha-fetoprotein (AFP)-containing hepatocytes in 12 human embryos and fetuses ranging from 30-32 days of estimated ovulation age (Streeter's horizon XIV) to 16-17 weeks of estimated menstrual age was investigated using the direct (horseradish peroxidase (HRP)-labeled anti-human AFP horse IgG(Fab')2) and/or indirect immunoperoxidase methods. Under light microscopy, the AFP-containing hepatocytes were successfully demonstrated in paraffin-embedded liver tissues fixed with 4% paraformaldehyde(PFA)-picric acid solution containing 0.5% glutaraldehyde (GA). As a result of the studies of the human fetal livers at different developmental stages, only a small number of AFP-containing hepatocytes were initially identified immunohistochemically at the stage of Streeter's horizon XIX. Ultrastructural immunohistochemistry (the block-staining method using anti-human AFP horse IgG(Fab')2) revealed that the immunoreactive products against AFP were demonstrated on the ribosomal granules of the rough endoplasmic reticulum (rER), outer nuclear envelopes, free ribosomes, and Golgi's apparatus, and also in the cisternal lumina of the ER. No reactive products were noted in the nuclei or mitochondria. Our observations confirmed the presence of AFP-producing ability of the hepatocytes and the ultrastructural localization of the sites of protein synthesis in the early stage of development of human livers. Furthermore, we describe the extreme usefulness of the block-staining method for demonstrating the subcellular localization of AFP using HRP-labeled reduced anti-AFP antisera on liver tissues fixed with 4% PFA-picric acid solution containing 0.5% GA.
The metabolism of benzo(a)pyrene (BP) in epidermal keratinocytes and dermal fibroblasts of humans and mice was investigated with emphasis on variation among species, individuals, and cell types. Human epidermal and dermal cells were isolated from the skin of normal subjects by trypsinization at 4 degrees overnight, followed by separation of the epidermis from the dermis with forceps. In confirmation of previous studies metabolic activity of human epidermal cells on BP was consistently demonstrated by cell-mediated assay, in which V79 Chinese hamster cells were plated on top of sheets of epidermal cells and treated with BP for 48 hr. Mutation of the V79 cells, measured as ouabain resistance, was induced in a dose-related fashion, although the extent of induced mutation varied from 5 to 22 ouabain-resistant colonies per 10(6) survivors/10 microM BP in cultures derived from different individuals. The most striking observation was that human dermal fibroblasts did not activate BP to a form that was mutagenic to cocultured V79 cells. This was observed without exception in all nine cultures of dermal fibroblasts and the one culture of embryo fibroblasts (MR-90) tested. Analysis by high-pressure liquid chromatography indicated that human epidermal and dermal cells both metabolized BP, producing almost the whole series of known metabolites of BP. The amount of BP 7, 8-dihydrodiol, a proximate metabolite of BP, produced by human dermal cells varied from 0.2 to 2.7% of the total BP added and seemed to be enough to induce mutation. Furthermore, human dermal cells not necessarily activated exogenously added BP 7,8-dihydrodiol to a form being mutagenic to V79 cells. These observations suggest that further metabolism of BP 7,8-dihydrodiol is partially or entirely blocked in human fibroblasts. In contrast to human fibroblasts, mouse fibroblasts isolated from the dermis of embryos did activate BP and induced mutation in cocultured V79 cells to a higher extent than did mouse epidermal cells, indicating interspecies variation in metabolic activation of BP between human and mouse fibroblasts.
The metabolism of benzo[a]pyrene (BP) and other carcinogens was investigated in primary cultures of mouse epidermal keratinocytes isolated from the C3H/He inbred strain, a responsive strain in the Ah locus. The basal aryl hydrocarbon hydroxylase activity of these cells was 9.2 pmol equivalents of 3-hydroxy-BP per mg protein per hr. This enzyme was induced dose-dependently by benz[a]anthracene (BA) and a 7-fold increase was obtained by treatment with 13 microM BA for 24 hr. The metabolism of BP increased with the time of incubation. On thin layer chromatography, 60% of the added BP was separated as metabolites at 24 hr and 96% at 48 hr. High pressure liquid chromatography indicated that mouse epidermal cells produced almost the whole series of metabolites of BP; dihydrodiols at the 9,10-,4,5- and 7,8-positions, quinones, and phenols at the 3- and 9-positions. The metabolic activity of mouse epidermal cells was further demonstrated by a cell-mediated assay, in which V79 Chinese hamster cells were cultured on top of epidermal cells and treated with BP and other carcinogens. Mutation of V79 cells, detected as ouabain resistance, was induced in a dose-related fashion by BP, BP-7,8-dihydrodiol, methylcholanthrene, and 7,12-dimethylbenz[a]anthracene, but not by aflatoxin B1; of these compounds BP-7,8-dihydrodiol was the most potent.
The effects of hemin on the metabolism of benzo[a]pyrene (BP) in rat liver microsomes were studied. Hemin inhibited the activity of aryl hydrocarbon hydroxylase and the metabolic activation of BP to forms able to bind to DNA. Analyses by high performance liquid chromatography of metabolites of BP revealed that hemin reduced their production and changed their relative amounts. The decrease in the amount of BP-7,8-dihydrodiol was greater than that in the amount of phenols. The elution profile of BP-metabolite-bound deoxyribonucleosides on Sephadex LH-20 column chromatography indicated that the formation of adducts with BP-7,8-dihydrodiol-9,10-oxide was decreased by hemin more than the formation of those with phenol derivatives. The present results suggest that endogenous components that are not enzymes are important modifiers of the biological activities of chemical carcinogens.
Differences in individual susceptibility to some kind of drug are mainly attributable to genetically determined differences in metabolic capability, although other factors, such as pathways of absorption, distribution and excretion, are important. A man, who has an impaired ability of the metabolism of a drug, responds sensitively to adverse effects of the drug. Recently, a development on assays of drug oxidation reactions shows that a frequency of occurrence in poor metabolizers in the population is observed differently in various ethnic groups. For example, in the metabolism of debrisoquine, an antihypertensive drug, the frequency in the British and the Nigerian is around 8-9%, while that in the Saudi Arabian and the Egyptian is 1%. To measure the oxidation status for debrisoquine is found to be useful tool for determining individual capability to oxidize various kinds of drugs. The cancer group contains a disproportionately large number of individuals who are extensive oxidizers compared to the control. Since most carcinogens require metabolic oxidation to generate reactive intemediates, which interact with nucleophiles in the cell, an assumption arises that there may be an association between genetically determined oxidation status and propensity to develop tumors in response to carcinogens in the environment.
In 97 nontoxic thyroid tumors, detection of thyroxine(T4) and triiodothyronine(T3) was attempted by the immunoperoxidase method. T4 was demonstrated in 58 tumors (59.8%) and T3 in 76 (78.4%). The feasibility of biosynthesis of T4 and T3 by such tumors was thus established. In 65 of the tumors, we applied the immunostaining method to serial or semiserial sections to study the correlation among the localizations of thyroglobulin(Tg), T4, and T3. The localization of T4 agreed relatively well with that of Tg, and part of the Tg-positive structure frequently revealed simultaneous positive staining for T4. The localization of T3, however, did not always correspond with that of T4 or Tg. T4 and T3 with localizations identical to that of Tg may be considered to be bound to Tg, but the mode of existence of T3 without correspondence in localization to Tg remains unknown.
Thyroid tissue specimens from 27 patients with thyroid tumors were examined for thyroxine (T4) and triiodothyronine (T3) by the peroxidase-labeled antibody method. The result revealed localization of T4 in 12 of the 14 follicular adenomas, in all the 8 papillary carcinomas and in 1 of the 3 follicular carcinomas studied, and of T3 in 13 of the 14 follicular adenomas, in all the 8 papillary carcinomas and in all the 3 follicular carcinomas. In the tumor tissue, the thyroid hormones were demonstrated in the colloid substance, on the luminal surface of tumor cells and in their cytoplasm. Compared with nontumorous thyroid tissue, the tumor tissue showed localization of the hormones predominantly in the cytoplasm and to a lesser extent in the colloid substance, with conspicuous variations in tissue distribution of positive areas and intensity of staining. This tendency was more marked in thyroid carcinomas. The demonstration of T4 and T3 in routine histological paraffin sections of formalin-fixed thyroid tissues in this investigation indicates potential usefulness of thyroid hormone detection by the peroxidase-labeled antibody technique. It is an effective diagnostic tool for evaluating the functional activity of the thyroid tumors as well as for determining whether a malignant growth under examination originates from the thyroid.
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