Formation Of Several Metabolites Research Articles

Metabolic re-programming in confrontations of Colletotrichum graminicola and Aspergillus nidulans with Bacillus biocontrol agents

We established confrontations between two different fungi, i.e., the maize anthracnose and stalk rot pathogen Colletotrichum graminicola, and the ubiquitous fungus Aspergillus nidulans, and different biocontrol species, i.e., Bacillus subtilis, Bacillus velezensis, and Bacillus amyloliquefaciens. In all fungus–bacterium confrontations tested, growth arrest and, thus, distance inhibition was observed on solid substrata. LC–MS/MS analyses of culture filtrates suggested formation of several metabolites only synthesized in confrontations. Interestingly, microscopy of fungal hyphae grown in liquid medium showed protrusions and color changes occurred only in media harboring fungus-bacterium confrontations. These observations indicate metabolic re-programming and suggest formation of putative secondary metabolites in interactions involving microbial biocontrol agents.

Biodegradation of low-density polyethylene plastic waste by a constructed tri-culture yeast consortium from wood-feeding termite: Degradation mechanism and pathway

Polyethylene (PE) is one of the most common synthetic polymers, and PE waste pollution has been an environmental and health concern for decades. Biodegradation is the most eco-friendly and effective approach for plastic waste management. Recently, an emphasis has been placed on novel symbiotic yeasts isolated from termite guts as promising microbiomes for multiple biotechnological applications. This study might be the first to explore the potential of a constructed tri-culture yeast consortium, designated as DYC, isolated from termites for the degradation of low-density polyethylene (LDPE). The yeast consortium DYC stands for the molecularly identified species Sterigmatomyces halophilus, Meyerozyma guilliermondii, and Meyerozyma caribbica. The LDPE-DYC consortium showed a high growth rate on UV-sterilized LDPE as a sole carbon source, resulting in a reduction in tensile strength (TS) of 63.4% and a net LDPE mass reduction of 33.2% compared to the individual yeasts. All yeasts, individually and in consortium, showed a high production rate for LDPE-degrading enzymes. The hypothetical LDPE biodegradation pathway that was proposed revealed the formation of several metabolites, including alkanes, aldehydes, ethanol, and fatty acids. This study emphasizes a novel concept for using LDPE-degrading yeasts from wood-feeding termites for plastic waste biodegradation.

Influence of the Photodegradation of Azathioprine on DNA and Cells.

Azathioprine (AZA) is a pharmacologic immunosuppressive agent administrated in various conditions such as autoimmune disease or to prevent the rejection of organ transplantation. The mechanism of action is based on its biologically active metabolite 6-mercaptopurine (6-MP), which is converted, among others, into thioguanine nucleotides capable of incorporating into replicating DNA, which may act as a strong UV chromophore and trigger DNA oxidation. The interaction between azathioprine and DNA, before and after exposure to solar simulator radiation, was investigated using UV-vis spectrometry and differential pulse voltammetry at a glassy carbon electrode. The results indicated that the interaction of AZA with UV radiation was pH-dependent and occurred with the formation of several metabolites, which induced oxidative damage in DNA, and the formation of DNA-metabolite adducts. Moreover, the viability assays obtained for the L929 cell culture showed that both azathioprine and degraded azathioprine induced a decrease in cell proliferation.

Effective Remediation Strategy for Xenobiotic Zoxamide by Pure Bacterial Strains, Escherichia coli, Streptococcus pyogenes, and Streptococcus pneumoniae.

Zoxamide, a class IV hazardous fungicide, is perilous for the environment due to its highly persistent nature. Up till the current date, there are no reports on the biodegradation of zoxamide. The scarcity of knowledge in this domain led to the present research to evaluate the biodegradation of this benzamide fungicide by three bacterial strains, Escherichia coli (EC), Streptococcus pyogenes (SPy), and Streptococcus pneumoniae (SP). Biotransformation of zoxamide was scrutinized in nutrient broth assemblies for a period of 28 days followed by UV-visible spectrophotometer and GC-MS analysis of the metabolites. The results exhibited a low to medium biodegradation potential of the bacterial cells to metabolize zoxamide. The highest biotransformation percentage was observed by E. coli to be 29.8%. The order of half-life calculated for the degradation results was EC (42.5) < SPy (58.7) < SP (67.9) days. GC-MS analysis indicated the formation of several metabolites including, 2-(3,5-dichloro-4-methylphenyl)-4-ethyl-4-methyl-4H-1,3-oxazin-5(6H)-one, 3,5-dichloro-N-(3-hydroxy-1-ethyl-1-methyl--2-oxopropyl)-4-methylbenzamide and 3,5-dichloro-4-methylbenzamide. The research could influence the biotreatment strategies for the environmentally friendly eradication of xenobiotics.

In vitro metabolism of sunscreen compounds by liquid chromatography/high-resolution tandem mass spectrometry.

Exposure to UV light can induce adverse effects on human health, such as photo-aging, immunosuppression, and cancer. Sunscreens are used to prevent the absorption of UV rays, but certain UV-filtering compounds have been shown to disrupt endocrine systems or act as carcinogens. To assess the effects of the exposure to such compounds, it is important to study the pathways by which they are biotransformed in the body. Liquid chromatography coupled to high-resolution tandem mass spectrometry (LC/HRMS/MS) was employed to evaluate the oxidative metabolism and, specifically, the formation of reactive metabolites of six active ingredients commonly used in sunscreen formulations: oxybenzone, avobenzone, homosalate, octisalate, octocrylene, and octinoxate. In vitro incubations were performed with human and rat liver microsomes in the presence of β-nicotinamide adenine dinucleotide phosphate and glutathione. An LC/HRMS/MS method was developed to identify metabolites employing a biphenyl reversed-phase column for separating parent molecules, metabolites, and glutathione (GSH) adducts. Each tested compound resulted in the formation of several metabolites, including at least one GSH adduct. Compounds containing ester groups were hydrolyzed, and some metabolites of the free acid forms were also detected. High-resolution MS/MS data was crucial for the structural elucidation of metabolites and GSH adducts. Fragmentation pathways were proposed for all parent compounds, as well as each described metabolite and adduct. The results of this study will help better understand the metabolism and detoxification pathways of these xenobiotics.

A Review on Role of Arecoline and Its Metabolites in the Molecular Pathogenesis of Oral Lesions with an Insight into Current Status of Its Metabolomics.

Areca nut consumption is a popular habit in Southeast Asian countries. One of the important biologically active alkaloids of areca nut is arecoline, which plays a role in mediating the development of several pathologies of the primary exposure site, the oral cavity. Studies on the metabolism of arecoline revealed the formation of several metabolites which themselves might be toxic. Moreover, polymorphisms in genes encoding enzymes involved in the metabolism of arecoline might predispose an organism towards the development of oral cancer. The present review tries to accumulate all the relevant existing literature and then elucidate the molecular mechanism by which arecoline plays a role in the development of oral submucous fibrosis and oral cancer. Existing information regarding arecoline metabolism, enzymes involved in the metabolic process and biological effects of the metabolites of arecoline have also been compiled and compared to study the toxicity of metabolites with its parent compound arecoline and whether they play any role in the pathogenesis of oral cancer mediated by areca nut consumption. A repertoire of molecular targets has come up in the discussion whose expression profile is perturbed by arecoline. Construction of induction cascade from existing literature has given an idea about the process of molecular pathogenesis. The summarized and analysed data can help to determine the molecular mechanism and drug targets, which in turn could be helpful in the prevention or treatment of these pathological conditions. It also brings into light areas where further research needs to be directed.

Bioactivation of the Cannabinoid Receptor Antagonist Rimonabant to a Cytotoxic Iminium Ion Metabolite

The cannabinoid type 1 receptor (CB1r) antagonist rimonabant was approved in 2006 for the treatment of obesity but was withdrawn in 2008 due to serious drug-related psychiatric disorders. In vitro metabolism studies with rimonabant have revealed high levels of reactive metabolite formation, which resulted in irreversible time-dependent P450 3A4 inhibition and in covalent binding to microsomal proteins. In the present study, an in vitro approach has been used to explore whether metabolic bioactivation of rimonabant might result in cell toxicity. A panel of SV40-T-antigen-immortalized human liver derived (THLE) cells that had been transfected with vectors encoding various human cytochrome P450 enzymes (THLE-1A2, 2C9, 2C19, 2D6, and 3A4) or with an empty vector (THLE-Null) were exposed to rimonabant. Cell toxicity and covalent binding to cellular proteins were evaluated, as was metabolite formation. Rimonabant exhibited markedly potentiated dose and time dependent cytotoxicity to THLE-3A4 cells, compared to that of all other THLE cell lines. This was accompanied by high levels of covalent binding of [(14)C]-rimonabant to THLE-3A4 cell proteins (1433 pmol drug equivalents/mg protein) and the formation of several metabolites that were not generated by THLE-Null cells. These included N-aminopiperidine (NAP) and an iminium ion species. However, no toxicity was observed when THLE cells were incubated with NAP. Glutathione depletion did not alter the observed potent cell cytotoxicity of rimonabant to THLE-3A4 cells. Preincubation of THLE-3A4 cells with the cytochrome P450 3A4 inhibitor ritonavir blocked the selective toxicity of rimonabant to these cells. In addition, ritonavir pretreatment blocked the metabolism of the compound in the cells and thereby significantly decreased the covalent binding of [(14)C]-rimonabant to THLE-3A4 cell proteins. We conclude that the potent toxicity of rimonabant in THLE-3A4 cells occurs by a mechanistic sequence, which is initiated by cytochrome P450 3A4 mediated formation of a highly cytotoxic reactive iminium ion metabolite that binds covalently to cellular proteins.

Isolation and characterization of homocholine-degrading Pseudomonas sp. strains A9 and B9b

Soil isolates, identified as Pseudomonas sp. strain A9 and Pseudomonas sp. strain B9b (based on the phenotypic features and phylogenetic analysis) were found to degrade homocholine aerobically. Morphological char- acterization using the optical microscope under light and phase contrast conditions showed that cells of strain A9 formed short rods measuring approximately 0.5-1 9 1.5- 2.0 lm in size while those of B9b formed long rods of 0.5-1 9 2.5-3.0 lm during the early growth phase on both nutrient broth and basal-homocholine (basal-HC) media. Strain A9 was able to grow on basal-HC medium at a wide range of temperatures (4-41C) whereas strain B9b was not able to grow at either 4 or 41C. Comparative 16S rRNA sequencing studies indicated that strain A9 fell into the Pseudomonas putida subclade whereas strain B9b located in Pseudomonas fulva subclade. Washed cells of strains A9 and B9b degraded homocholine completely within 6 h with concomitant formation of several metabolites. Analysis of the metabolites by capillary electrophoresis, fast atom bombardment-mass spectrometry, and gas chromatogra- phy-mass spectrometry, showed trimethylamine (TMA) as the major metabolite beside b-alanine betaine and tri- methylaminopropionaldehyde. Therefore, the possible degradation pathway of homocholine in the isolated strains is through successive oxidation of the alcohol group (-OH) to aldehyde (-CHO) and acid (-COOH), and thereafter the cleavage of b-alanine betaine C-N bonds yielding tri- methylamine and an alkyl chain.

Isolation and characterization of 3-N-trimethylamino-1-propanol-degradingRhodococcussp. strain A2

The aerobic degradation of 3-N-trimethylamino-1-propanol (homocholine) as a sole source of carbon and nitrogen has been found for a Rhodococcus sp. bacterium isolated from soil. The isolate was identified as Rhodococcus sp. strain A2 based on its phenotypic features, physiological and biochemical characteristics, and results of phylogenetic analysis. The washed cells of strain A2 completely degraded homocholine within 6 h, with concomitant formation of several metabolites. Analysis of the metabolites using capillary electrophoresis, fast atom bombardment-MS, and GC-MS showed that trimethylamine was the major metabolite, in addition to beta-alanine betaine (beta-AB) and trimethylaminopropionaldehyde. Therefore, the possible degradation pathway of homocholine in the isolated strain is through consequent oxidation of the alcohol group (-OH) to aldehyde (-CHO) and acid (-COOH). Thereafter, the cleavage of beta-AB C-N bonds yielded trimethylamine and alkyl chain.

Biodegradation of polyalcohol ethoxylate by a wastewater microbial consortium

Polyalcohol ethoxylate (PAE), an anionic surfactant, is the primary component in most laundry and dish wash detergents and is therefore highly loaded in domestic wastewater. Its biodegradation results in the formation of several metabolites and the fate of these metabolites through wastewater treatment plants, graywater recycling processes, and in the environment must be clearly understood. Biodegradation pathways for PAE were investigated in this project with a municipal wastewater microbial consortium. A microtiter-based oxygen sensor system was utilized to determine the preferential use of potential biodegradation products. Results show that while polyethylene glycols (PEGs) were readily degraded by PAE acclimated microorganisms, most of the carboxylic acids tested were not degraded. Biodegradation of PEGs suggests that hydrophobe-hydrophile scission was the dominant pathway for PAE biodegradation in this wastewater community. Ethylene glycol (EG) and diethylene glycol (DEG) were not utilized by microbial populations capable of degrading higher molecular weight EGs. It is possible that EG and DEG may accumulate. The microtiter-based oxygen sensor system was successfully utilized to elucidate information on PAE biodegradation pathways and could be applied to study biodegradation pathways for other important contaminants.

Study on the Decomposition of 2‐Chloroethylnitro‐ sosulfamides (CENS) in Serum Using HPLC On‐line Solid Phase Extraction

In this paper we have investigated the kinetic decomposition of 2-chloroethylnitrososulfamides (CENS) in fetal calf serum. The study was monitored by on-line solid phase extraction linked to RP-LC-MS. We demonstrated that CENS are less stable in fetal calf serum than in aqueous buffer at pH 7.4 and 37 degrees C. Moreover, we have shown that partition coefficient of CENS can be correlated to the kinetics decomposition, and we observe that the stability is better for lipophilic CENS. The different metabolites after decomposition have been tentatively identified by RP-HPLC-MS. This study indicates the formation of several metabolites resulting from a mechanism of decomposition more complex than in aqueous phosphate buffer, nevertheless, leading to the same main products.

First isolation and characterization of a bacterial strain that biotransforms the herbicide mesotrione

The aim of this study was to find and characterize a fungal or bacterial strain capable of metabolizing mesotrione, a new selective herbicide for control of broad-leaved weeds in maize. This strain was isolated from cloud water and showed close phylogenetic relationship with strains belonging to the Bacillus genus, based on 16S rRNA gene alignment. Kinetics of mesotrione degradation were monitored by high-performance liquid chromatography and in situ(1)H nuclear magnetic resonance spectroscopy at different concentrations. Mesotrione was completely biotransformed even at 5 mmol l(-1) concentration. 2-Amino-4-methylsulfonyl benzoic acid (AMBA) was identified as one of the metabolites, but was not the major one. This study reports the first rapid mesotrione biotransformation by a pure bacterial strain and the formation of several metabolites including AMBA. This bacterium isolated from cloud water is the first pure strain capable of rapidly degrading mesotrione.

In vitro metabolism of the mammalian soluble epoxide hydrolase inhibitor, 1-cyclohexyl-3-dodecyl-urea.

The metabolism of the soluble epoxide hydrolase (sEH) inhibitor, 1-cyclohexyl-3-dodecyl-urea (CDU), was studied in rat and human hepatic microsomes. The microsomal metabolism of CDU enhanced sEH inhibition potency of the reaction mixture and resulted in the formation of several metabolites. During the course of this study, a sensitive and specific high-performance liquid chromatography with tandem mass spectrometry analytical method was developed to investigate simultaneously the production of these metabolites. In both rat and human hepatic microsomes, CDU was ultimately transformed into the corresponding omega-carboxylate; however, the rodent tissue appeared to perform this transformation more rapidly. After a 60-min incubation in rat hepatic microsomes, the percentage of residual CDU, the omega-carboxylate, and the intermediary omega-hydroxyl were about 20%, 20%, and 50%, respectively. Carbon monoxide inhibited the metabolism of CDU by rat hepatic microsomes, suggesting that the initial step is catalyzed by cytochrome P450. Further metabolism was enhanced by the addition of NAD, suggesting that dehydrogenases are associated with intermediate metabolic steps. Regardless, the ultimate product of microsomal metabolism, 12-(3-cyclohexyl-ureido)-dodecanoic acid, is also an excellent sEH inhibitor with several hundred-fold higher solubility, supporting the hypothesis that CDU has prodrug characteristics. These findings will facilitate the rational design and optimization of sEH inhibitors with better physical properties and improved metabolic stability.

Investigation on the transformation of fluchloralin by soil fungi: Identification of some metabolites

AbstractFluchloralin [N‐(2‐chloroethyl)‐α,α,α,‐trifluoro‐2,6‐dinitro‐N‐propyl‐p‐toluidine] was transformed by two common soil fungi, Aspergillus flavus Link and Fusarium solani (Mart.) Sacc. in pure culture. The fungi were isolated from enrichment cultures of an analogous herbicidal compound, pendimethalin [N‐(1‐ethylpropyl)‐2,6‐dinitro‐3,4‐xylidine)]. Transformation of fluchloralin by these fungi resulted in the formation of several metabolites, 13 of which were identified. Besides the known mechanisms of microbial transformation of dinitroanilines, methylation of the anilino nitrogen and direct elimination of a nitro group at 2 position from the aromatic ring without any further substitution were also observed.

The role of sulfation and/or acetylation in the metabolism of the cooked-food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in Salmonella typhimurium and isolated rat hepatocytes.

Mutagenic activity of the cooked-food mutagen/carcinogen 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) is highly dependent upon cytochrome P450 activation to the N-hydroxylated intermediate. In the present study the bioactivation pathways of PhIP were investigated in Salmonella typhimurium and isolated rat hepatocyte preparations. In the Ames/S. typhimurium assay, the acetyltransferase and sulfotransferase enzyme inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP) were used to modulate mutagenicity. DCNP, but not PCP, produced a concentration-dependent decrease in mutagenic activity of 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP). In rat hepatocyte preparations, PCP and DCNP, as well as the cytochrome P450 IA1 and IA2 inhibitor alpha-naphthoflavone (ANF), were used to modulate metabolite, protein adduct, and DNA adduct formation. Incubations of [3H]PhIP (100 microM) with Aroclor 1254-induced or uninduced hepatocytes resulted in the formation of several metabolites, including 4'-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate (4'-PhIP-sulfate), 2-amino-1-methyl-4'-hydroxy-6- phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP), a glucuronide conjugate of 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine, and other uncharacterized metabolites. While PCP or DCNP pretreatment produced a significant decline in sulfate-dependent conjugation of 4'-hydroxy-PhIP to 4'-PhIP-sulfate, these inhibitors produced only slight decreases in PhIP-dependent covalent binding to proteins in hepatocytes derived from either Aroclor 1254-induced or uninduced rats. PhIP DNA adduct levels were relatively unchanged by PCP or DCNP pretreatment of Aroclor 1254-induced hepatocytes. DNA adducts from hepatocytes dosed with N-hydroxy-PhIP, however, resulted in a decrease in adduct levels from cells pretreated with PCP or DCNP.(ABSTRACT TRUNCATED AT 250 WORDS)

Maintenance of the biotransformation capacity by cultured human hepatocytes after several daily exposures to drugs

Adult human hepatocytes were obtained from four different donors and exposed to three new drugs (DPI 201-106, CK 204-933 and SDZ 203-150) for 4 or 5 days with daily renewal of both medium and drug. Both the rates and the routes of metabolism were analysed every day by HPLC and thin-layer chromatography. Metabolism of the three drugs resulted in the formation of several metabolites, which involved various phase I and phase II reactions. For the three drugs, both qualitative and quantitative metabolic capacity was roughly maintained during the period studied. However, some minor metabolites were identified only after two or three incubations, probably because of their low rate of formation and/or multistep synthesis. Individual variations, which were maintained during the whole incubation period, were observed. The maintenance of the same metabolic pathways even after several daily treatments indicates that adult human hepatocytes in primary culture represent a suitable in vitro model system for studies on drug inhibition or induction, drug interactions and long-term drug cytotoxicity.

Metabolism of aflatoxin B1 in the bovine olfactory mucosa

Carcinomas of the ethmoidal region of the nose are observed relatively frequently in cattle in several countries in tropical and subtropical latitudes. Viruses have been implicated as causative agents, but it has been observed that affected animals sometimes suffer from aflatoxicosis, and a role of aflatoxin B1 (AFB1) in the aetiology has also been proposed. We have examined whether the bovine nasal olfactory mucosa has a capacity to metabolize AFB1. The contents of cytochrome P-450 and cytochrome b5, and the NADPH cytochrome c reductase activity in the nasal olfactory mucosa have also been determined. Comparative experiments have been performed with the liver. Incubations with 3H-labelled AFB1 showed that the nasal olfactory mucosa has a much higher capacity than the liver to form lipid-soluble, water-soluble and tissue-bound AFB1-metabolites. High-resolution microautoradiography showed a strong localization of tissue-bound metabolites in the sustentacular cells in the apical portion of the olfactory surface epithelium and in Bowman's glands in the olfactory lamina propria mucosae. Especially in the sustentacular cells the labelling was preferentially located in the nuclei of the cells. Liquid chromatography of chloroform extracts of the nasal olfactory mucosa and the liver incubated with 3H-AFB1 showed formation of several metabolites. The dominating peak in both tissues was aflatoxin M1 (AFM1). However, the amount of AFM1 was higher in the nasal olfactory mucosa than in the liver, and the amounts and proportions of several other metabolites also differed markedly between the two tissues. The level of cytochrome P-450 in the nasal olfactory mucosa was found to be about one quarter of that in the liver, but the NADPH cytochrome c reductase activity was much higher in the nasal olfactory mucosa than in the liver. In addition, the cytochrome b5: cytochrome P-450 ratio was higher in the nasal olfactory mucosa than in the liver. The higher metabolism of AFB1 in the nasal olfactory mucosa than in the liver may be related to differences in the cytochrome P-450 isoenzyme profile. In addition, the microsomal electron transport to cytochrome P-450 may be facilitated by the high reductase: cytochrome P-450 ratio and the high cytochrome b5: cytochrome P-450 ratio in the nasal olfactory mucosa.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of lonidamine on the utilization of 14C-labeled glucose by human astrocytoma cells

The effect of lonidamine (LND), 1-(2,4-dichlorobenzyl)-1 H-indazol-3 carboxylic acid, on the utilization of carbon from 14C-labeled glucose by cell cultures of the permanent strain LI derived from a human glioblastoma multiforme (astrocytoma) has been investigated. The results may be summarized as follows. Aerobic glycolysis is the main energy-yielding process as shown by the fact that the greatest part of glucose carbon atoms is incorporated into lactate. Nevertheless, the amount of glucose converted accounts for only 63% of the lactate produced, indicating the presence of an elevated endogenous aerobic glycolysis. The amount of glucose carbon atoms incorporated into CO 2, lipids, nucleic acid, and supporting structures is low. LND decreases the incorporation of 14C activity in all the above mentioned isolated compounds because of its ability to inhibit glucose phosphorylation. Consequently, there is a lower concentration of glucose-6-phosphate which, in turn, affects the rate of formation of several metabolites in glycolytic and pentose phosphate pathways. Experiments with [1- 14C]-2-deoxy- d-glucose further substantiate the idea of glucose phosphorylation as a main target of LND and strongly suggest the presence of a mitochondrially bound hexokinase. The higher inhibition of glucose phosphorylation in exponentially growing cells indicates a further shift of the enzyme toward mitochondria-bound form and confirms the importance of the energy status of the cell in eliciting the response to LND. The reduced capacity of LND-treated cells to synthetize ATP and glucose-6-phosphate reflects the decreased synthesis of proteins and nucleic acids, which affects cell growth and duplication.

Metabolic activation of 3-nitroperylene in the Salmonella/S9 assay.

The mutagenicity of 3-nitroperylene (nitroPer), a nitrated polycyclic aromatic hydrocarbon (PAH) has been extensively studied using the Ames Salmonella test. In accord with previous work, nitroPer proved to be a potent frameshift mutagen which requires activation by mixed function oxidases (MFOs) in the microsomes of rat liver S9 preparations. The concentration of S9 required for optimal activation of this mutagen is several times lower than that recommended for routine screening by the Ames test. Studies with the MFO inducers Aroclor 1254, phenobarbital, 3-methylcholanthrene and beta-naphthoflavone, as well as with some MFO inhibitors indicate that both P450b and P450c appear to be involved in the activation of nitroPer. Two non-mutagenic PAHs (perylene and benzo[e]pyrene) inhibited the mutagenicity of nitroPer in a competitive fashion. The mutagenic activity of nitroPer was greatly decreased in a strain of bacteria (TA98/1,8-DNP6) that lacks an acetyltransferase needed for the activation of many nitroarenes. Incubation of nitroPer with microsomes from Aroclor-treated rats plus appropriate cofactors led to the formation of several metabolites which could be separated from one another and from nitroPer by h.p.l.c. Three of these were direct-acting mutagens with Salmonella typhimurium strain TA98, while another required microsomal activation. We postulate that the metabolic activation of nitroPer requires three steps: (i) metabolism by MOF enzymes to yield a ring-oxidized compound which is absorbed by the bacteria; (ii) reduction of this compound to the hydroxylamine by a bacterial nitroreductase; and (iii) O-acetylation of the hydroxylamine to yield a reactive ultimate mutagen.

15] A new pathway of 25-hydroxyvitamin D3 metabolism

This chapter demonstrates the in vitro formation of several metabolites of 25-hydroxyvitamin D 3 (25- OHD 3 ), which can be logically placed on a single metabolic pathway, using the isolated perfused rat kidney. Each metabolite can be identified by a combination of high-performance liquid chromatography (HPLC), ultraviolet (UV) spectrophotometric, and mass spectrometric techniques. The enzymes involved in the pathway are present only in the vitamin D-repleted animal, are stimulated by vitamin D intoxication, and probably play some role in excretion of the potentially toxic molecule, 25-OHD 3 . The study of the pathway of nonradioactive 25-OHD 3 metabolism in preparative sized samples requires a different approach that involves three steps from bulk extract to pure material suitable for mass spectrometry, and a third step back on Zorbax-SIL with the initial solvent to confirm purity. Such a scheme separates most of the metabolites of 25-OHD 3 based upon their hydroxyl content while avoiding the column overloading problems encountered with large lipid contents of bulk perfusate extracts. The chapter also discusses the diode-array spectrophotometric detection of metabolites of 25-OHD 3 and identification of metabolites of pathway. The metabolite 24,25-(OH) 2 D 3 has been known as an in vivo and in vitro product of 25-OHD 3 metabolism. Using 24,25-(OH) 2 D 3 as a substrate for the perfused rat kidney it is shown that the three metabolites described in the chapter appear in the perfusate within 2-4 hr. If a vitamin D-depleted donor rat is used, no such metabolites are formed from 24,25-(OH) 2 D 3 until 4-6 hr has elapsed. Use of 24-oxo-25-OHD 3 as substrate in the vitamin D-repleted kidney results in synthesis of 24-oxo-23,25-(OH) 2 D 3 and 24,25,26,27-tetranor-23-OHD 3 but not 24,25-(OH) 2 D 3 . Use of 23(S),25- (OH) 2 D 3 gives only 25-OHD 3 -26,23-lactone. Use of 24-oxo-23,25-(OH) 2 D 3 as substrate in the vitamin D-repleted kidney results in synthesis of only 24,25,26,27-tetranor-23-OHD 3 .