Presence Of Microsomal Enzymes Research Articles

COMPUTATIONAL APPROACHES RELATED TO DRUG DISPOSITION

Drug disposition connects with the movement of drug molecules inside the body after administration irrespective with the route of administration. After entering the system, drug molecule and internal body systems comes under various pharmacokinetic interactions followed by observation of suitable biological activity. In this exhaustive process, physicochemical nature of the chemical substance and physiological nature of system makes this movement competitive. In this view, pharmacokinetic and toxic properties of the molecule regulates the destination of the molecule. Various computational processes are available for in silico pharmacokinetic assessment of drug molecule after absorption through biological membrane, distributed throughout the system based on the percent ionization or partition coefficient factors followed by biologically transformed into an another entity in presence of microsomal enzymes and finally excrete out from system using various cellular transport systems as well as related cellular toxicity behavior. In this chapter, we ensemble all the possible information related with the drug movement and related computational tools to understand the possible chemical and pathophysiological changes. Here detailed knowledge on database expedition, establishment of pharmacophore model, homology modelling based on sequence similarity, molecular docking study (rigid and flexible docking) and QSAR/QSPR study (with detailed process and available softwares) are provided. These diversely united informations actually helps a researcher to understand the factual movement of a drug molecule inside the system.

Redox Cycling of 2-(x′-Mono, -di, -trichlorophenyl)- 1,4-benzoquinones, Oxidation Products of Polychlorinated Biphenyls

Polychlorinated biphenyl (PCB) preparations are complete liver carcinogens in rodents and efficacious promoters in two-stage hepatocarcinogenesis. Cytochrome P450 isozymes catalyze the oxidation of PCBs to mono- and dihydroxy metabolites. The potential for further enzymatic or nonenzymatic oxidation of ortho- and para-dihydroxy PCB metabolites to (semi)quinones raises the possibility that redox cycling involving reactive oxygen species may be involved in PCB toxicity. Seven synthetic 2-(x′-chlorophenyl)-1,4-benzoquinones (containing one to three chlorines) were investigated for their participation in oxidation–reduction reactions by following the oxidation of NADPH. These observations were made: (i) NADPH alone directly reduced all quinones but only 2-(2′-chlorophenyl)- and 2-(4′-chlorophenyl)-1,4-benzoquinone supported NADPH consumption beyond that required to quantitatively reduce the quinone. (ii) For all quinones, superoxide dismutase increased NADPH oxidation in excess of the amount of quinone, demonstrating the participation of the superoxide radical. (iii) The presence of microsomal enzymes from rat liver increased the rate of NADPH consumption, but only 2-(2′-chlorophenyl)- and 2-(4′-chlorophenyl)-1,4-benzoquinone autoxidized. (iv) The combination of superoxide dismutase with microsomal enzymes accelerated autoxidation from 1.6- to 6.8-fold higher than that found in the absence of microsomal protein. These data support the concept that in the absence of microsomal protein, there occurs a two-electron reduction of the quinone by NADPH to the corresponding hydroquinone that comproportionates with the large reservoir of quinone to initiate autoxidation. In the presence of microsomes, enzymatic one-electron reduction generates a semiquinone radical whose autoxidation with oxygen propagates the redox cycle. These results show the potential of some 2-(x′-chlorophenyl)-1,4-benzoquinones to initiate the wasteful loss of NADPH.

Influence of metabolic genotype GSTM1 on levels of urinary mutagens in patients treated topically with coal tar

Fifteen hospitalized, non-smoking, dermatological patients were treated with ointment containing 2% coal tar (CT) in order to assess the influence of metabolic genotype GSTM1 on urinary mutagen levels. Urinary 1-pyrenol, the main metabolite of pyrene, was used to check the high exposure to PAH of this population. The mean levels of urinary 1-pyrenol found in the 24-h urine of our patients were 467.8±211.0 nmoles-24 h (range 94.6–890.1 nmoles-24 h). Mutagenicity was assessed on urine samples collected over a period of 24 h, after three consecutive days of topical application, using the bacterial mutagenesis test on Salmonella typhimurium strains TA98 and YG1024 in the presence of microsomal enzymes. The latter strain turned out to be more sensitive than the former in revealing urinary mutagens in these patients (42 693±30 867 vs. 6877±6040 net revertants-24 h). The mutagenicity on YG1024 strain and 1-pyrenol levels of urine samples were correlated (Spearman's rank correlation coefficient=0.6678, P<0.01, z=2.795). The influence of genotype GSTM1 on urinary mutagen levels was assessed on strain YG1024. The values of urinary mutagenicity of subjects with genotype GSTM1-null ( n=6) were on average higher than those of GSTM1-positive subjects ( n=9) (55 498±45 957 vs. 34 156±11 933 net rev.-24 h), a non-significant statistical difference. The mean total excretion of mutagens corrected for PAH exposure (net rev./nmoles of urinary 1-pyrenol) in GSTM1-null patients was double that of GSTM1-positive ones (136.8±34.7 vs. 70.8±23.3 net rev./nmoles of urinary 1-pyrenol; one-tailed Mann–Whitney U-test, U=11.5, P<0.05). These results indicate a greater body burden of promutagens, resulting from skin application of CT, in GSTM1-null subjects.

The mutagenicity of edible mushrooms in a histidine-independent bacterial test system

In mutagenicity screening of 40 edible mushroom species, special attention was paid to the selection of the test system, since complex mixtures such as mushroom extracts may interfere with the genetic endpoint of the assay. This paper shows that the weak mutagenicity of some mushrooms in the Ames test, as reported by several authors, is actually an artefact due to the presence of free histidine in the mushroom extracts, which apparently increases the reversion of bacteria from histidine auxotrophy to prototrophy. To avoid amino-acid interaction, a combination of the forward mutation assay to 8-azaguanine resistance in Salmonella typhimurium TM677 and a liquid test was used. Out of 35 wild and commercially grown mushrooms tested, 13 species exhibited mutagenic activity. In the case of the five samples of dried mushrooms, weak mutagenicity could be detected for Auricularia sp. The presence of microsomal enzymes (S-9) reduced the mutagenic effects of all the mushrooms, with the exception of Agaricus abruptibulbus and Cantharellus cibarius, where metabolic activation enhanced the mutagenic activity.

Interferon induction inhibition and mutagenic activity of nitrosated coal dust extract

Specified cytotoxicity and mutagenicity of coal dust extract (mixture of solvent extractions of bituminous coal nitrosated by NaNO2) were investigated because of the association of an excess risk of gastric cancer in coal miners. The effect of nitrosated coal dust on a cellular defense component of the interferon system, the induction of interferon (alpha/beta) in mammalian cell cultures by influenza virus, and mutagenicity, using the Salmonella/microsome assay, were determined. Nitrosated coal dust extract contained both bioactivation-independent and -dependent (microsome enzymatic activation) compounds that significantly inhibited the viral induction of interferon by greater than 50%. Nitrosated extract was mutagenic but exhibited no increase in mutagenic activity in the presence of microsomal enzymes. With further extraction of nitrosated coal dust extract by horse serum and fractionation thereof, the soluble chemical complexes formed with fractions of high molecular weight without bioactivation were dominant in both mutagenicity and inhibition of interferon induction. Low-molecular-weight fractions, with or without a metal chelator, and with or without bioactivation, all inhibited interferon induction comparably and significantly. There was no mutagenic activity manifested by these serum fractions. Metal-serum complexes were either not formed, or, if present, were ineffectual according to the biologic criteria employed. The findings of this study are discussed in terms of the association between nitrosated coal dust and gastric carcinogenesis.

The initiator tRNA acceptance assay as a short-term test for carcinogens. 1. A standardized procedure.

A short-term test for carcinogens has been developed based on the interaction of chemical carcinogens with tRNA(FMet) in vitro. Transfer RNA from rat or rabbit liver is pre-treated with compounds to be tested in the presence of microsomal enzymes and NADPH. Re-isolated tRNA is then charged with L-methionine by aminoacyl-tRNA synthetases from E. coli B. Carcinogens induce a stimulation of tRNA charging whereas chemically similar non-carcinogenic compounds do not show this effect. Experiments with model substances N-methyl-N'-nitro-N-nitrosoguanidine (strong carcinogen) and aflatoxin G1 (weak carcinogen) revealed some differences in dose effect relationships. It is advisable to test unknown compounds at three different concentrations (10(-5), 10(-7) and 10(-9) mg/ml) with at least two different quantities of microsomal enzymes. Tests on greater than 150 different compounds performed so far indicate that the evaluation of results as % of stimulation (when compared with the control value obtained with the charging of tRNA treated with the solvent only) may allow a quantitative discrimination between weak and intermediate, and strong carcinogens. The procedure is rapid, well reproducible and relatively inexpensive and may be used to complement the other short-term tests for carcinogenicity.

Microsome-mediated stimulation of the aminoacylation of rat liver initiator transfer ribonucleic acid by benzo[ a]pyrene and 3,3-dimethyl-1-phenyltriazene

The charging of initiator tRNA F met with L-methionine was significantly stimulated by pre-incubation of unfractionated tRNA from rat liver with benzo[ a]pyrene (BP) or 3,3-dimethyl-1-phenyltriazene (DMPT). The presence of microsomal enzymes from rat liver and of NADPH was absolutely required for this effect. Maximum enhancement was obtained after 60 min of incubation with 10 −4–10 −3 μmol/ml of either compound tested. It appears that either proximate carcinogen, BP or DMPT, must be converted by microsomal enzymes into its direct-acting metabolites (ultimate carcinogens) which interact with initiator tRNA and thereby specifically modulate its aminoacylation.

Microsome-dependent binding of benzo[ a]pyrene to initiator tRNA from rat liver

[ 3H]Benzo[ a]pyrene (BP) becomes bound to initiator tRNA from rat liver it incubated with it in the presence of microsomal enzymes and NADPH. The reaction proceeds linearly for 30 min at 37°C and is stimulated by MgCl 2 (1–2 mM) and KCl (80–100 mM). BP must be apparently converted to its active metabolites (ultimate carcinogens) by microsomal enzymes and these metabolites are then bound to tRNA F Met.

Mutagens in larger fungi: I. Forty-eight species screened for mutagenic activity in the Salmonella/microsome assay

Specimens of large fungi (mushrooms) were screened for mutagenic activity by the Salmonella/microsome assay, with strains TA98, TA2637 and TA100. Out of 48 species tested, 37 exhibited a significant but for the most part weak activity. The activity observed in the presence of S9 mix was typically between 0 and 50% of that without, and in no case was the activity increased in the presence of microsomal enzymes. Six metabolites reported to occur in some of the species included in this investigation were also tested. Significant mutagenic activity was found with isovelleral ( 5) from Lactarius sp., agaritine ( 3) from Agaricus bisporus and related sp. and β-nitraminoalanine ( 7) from Agaricus silvaticus. Isovelleral may be a major mutagen in some of the sharp-tasting and mutagenic Russulaceae sp. A. bisporus (cultivated specimen) was weakly mutagenic toward all three strains of S. typhimurium used, and agaritine was weakly active toward TA2637 alone. This implies that this fungus might contain other mutagenic material as well. β-Nitraminoalanine was not found in the particular collection of A. silvaticus tested here. The mutagenicity observed for the fungus in this work may therefore be due to other metabolites. Even though many species found to be mutagenic are used as food, it seems premature to make specific recommendations about eventual health risks. Further information is needed about the chemistry and toxicology of the active compounds as well as about the effects of various methods used in preparing mushrooms for food.

Proflavin: an unusual mutagen

In the Salmonella typhimurium mutagenicity assay, proflavin (PF), in the dark, was shown to possess some direct-acting frameshift mutagenic activity for strains TA1537 and TA98. In the presence of microsomal enzymes from mouse, hamster and rat livers, frameshift mutagenic activity for strain TA1538 and enhanced mutagenicity for strain TA1537 was observed. Exposure of bacteria to PF in the presence of visible light resulted in base substitution (strain TA1535) as well as frameshift (TA1537, TA1538, TA98) genetic activities. The findings are taken to indicate that PF possesses direct-acting frameshift activity (TA1537 and TA98) by virtue of its ability to intercalate between DNA basepairs. The frameshift activity (TA1538, TA98 and enhanced TA1537) seen following metabolic or photo-activation may be due to the formation of chemical adducts between the cellular DNA and oxidation products of PF (a base-displacement effect). The light-induced base-substitution activity probably results from the generation of singlet oxygen which reacts with the guanine moiety of DNA. The mutagenic photoproduct of PF appears to be stable.

Mutagenic evaluation of nitroparaffins in the salmonella typhimurium/mammalian‐microsome test and the micronucleus test

Three nitroparaffins (nitroethane, 1-nitropropane, and 2-nitropropane) were studied in the Salmonella typhimurium/mammalian microsome (Ames) test, with and without microsomal activation systems. Nitroethane and 2-nitropropane also were studied in an in vivo mutagenic (micronucleus) test. These studies were undertaken because these solvents are widely used in the chemical and pharmaceutical industries and 2-nitropropane was reported to cause liver cancer in rats exposed by the inhalation route. Neither nitroethane nor 1-nitropropane was active in the Ames test with Salmonella tester-strains TA1537, TA92, TA98, or TA100. However, 2-nitropropane produced a significant increase in revertants in all of these tester strains, particularly strain TA100, where 3 microliter/plate doubled the number of revertants in the presence of microsomal enzymes. Negative results were obtained with both nitroethane and 2-nitropropane in micronucleus tests. These studies have shown that 2-nitropropane has the potential for causing point mutations in a microbial test system. However, this compound probably will not cause a chromosome mutation of the clastogenic type.

Enhancement of 5-iododeoxyuridine-induced endogenous C-type virus activation by polycyclic hydrocarbons: apparent lack of parallelism between enhancement and carcinogenicity.

When mouse MLg cells were treated with 3-methylcholanthrene or 7,12-dimethylbenz[alpha]anthracene in the presence of microsomal enzymes and NADPH after 5-iododeoxyuridine (IUDR) treatment, the induction rate of the endogenous C-type virus was increased fivefold to sixfold in comparison with the culture treated with IUDR only. In this reaction, both the microsomal enzymes and NADPH were indispensable. 7,8-Benzoflavone, an inhibitor of the metabolism of hydrocarbons in hamster embryo cultures, inhibited the reaction. For detecting the enhancing activity, the concentration of IUDR for the pretreatment, the concentration of the test products, and the duration of the treatment with the products were important factors. In screening 30 polycyclic hydrocarbons, we were unable to detect a correlation between the in vivo carcinogenicity in the skin and the enhancing activity in the conditions tested.

Cytotoxic activity of in vitro activated cyclophosphamide on tumors in primary culture.

An in vitro method for testing the cytotoxic activity of compounds active through the formation of metabolites is described, and cyclophosphamide (CPA) has been chosen as a « model » substance. A tumor primary explant, cultured in a Rose chamber, was treated for 90 min at 37 °C with CPA in the presence of microsomal enzymes (105,000 × g liver fraction) and their cofactors. At the end of the incubation, the medium was removed and changed. Appropriate controls are concurrently carried out through the procedure. The cytotoxic effect of CPA is evaluated 24 h after the treatment. Primary explants from both mammary tumors of (C3H × O20)F1 mice and human ovary tumors were sensitive to CPA under our experimental conditions. The significance of this procedure for in vitro characterization of tumor sensitivity to antineoplastic agents which requires hepatic biotransformation is emphasized.