Liver Microsomal Fractions Research Articles

Characterization and estimation of sulfaquinoxaline metabolites in animal tissues using liquid chromatography coupled to tandem mass spectrometry

Sulfaquinoxaline (SQX) is a sulfonamide that is widely used in veterinary medicine, with a maximum residue limit (MRL) established for several food matrices. In Brazil, the MRL for liver and muscle is 100 μg kg−1 for equine, bovine, poultry and swine. This value includes not only free drug but also the sum of all metabolites. Several reports showed limitations for SQX residue analysis, especially when mass spectrometry methods were used. These limitations include poor recoveries and unacceptable accuracy responses. In this work a metabolite of SQX, present in liver and kidney samples, was identified. The structure proposed was a hydroxylated form of SQX, called SQX-OH, with an m/z of 317. SQX-OH is also produced in vitro in equine, swine and bovine liver samples. The influence of time, temperature, solvent and dehydration was evaluated in the formation of SQX-OH. Different degrees of hydroxylation were observed in matrices. The N4-acetyl derivates for both SQX and SQX-OH were also detected. In equines, the metabolism of SQX is complete. The mass spectrometry analysis of SQX-OH was determined in vitro using equine liver microsomal fraction. The characterization of this compound was performed using liquid chromatography coupled to mass spectrometry in tandem mode. The fragmentation profile of SQX-OH was seen to be similar to that of the sulfonamides group, producing two high abundant daughter ions: 317 > 156 and 317 > 108, common to most sulfonamides. The main conclusion of this work is that the residue analysis of SQX needs to consider the presence of the SQX-OH in order to give more realistic results, especially when using MRM transitions.

Optimization of the SDS-PAGE gel slicing approach for identification of human liver microsomal proteins via MALDI-TOF mass spectrometry

Proteomics often exploits sequential cutting of the SDS-PAGE gel lane into slices with subsequent identification of proteins by peptide mass fingerprinting (PMF). In this paper, the influence of slice thickness on protein identification was investigated. Following the separation of the human liver microsomal fraction, the 37 to 75 kDa range of the gel lane was cut into 20 or 40 slices of 0.5 mm and 0.25 mm thickness, respectively. Identification of proteins after trypsinolysis was performed by MALDI-TOF mass spectrometry. A twofold reduction of slice thickness did not influence the number of peaks in PMF-spectra. It was established that at a tolerance of 0.15 Da the number of identified proteins in the 40 slices series exceeded by more than twofold the number of identifications in 20 slices series. It was shown that the decrease of slice thickness leads to a considerable enhancement of peptide peak’s intensity and, also, to the changes of PMF-spectra which resulted gaining additional peptides for enrichment of sequence coverage.

Synthesis and Biological Activities of 1α,4α,25- and 1α,4β,25-Trihydroxyvitamin D<sub>3</sub> and Their Metabolism by Human CYP24A1 and UDP-Glucuronosyltransferase

A previous report has demonstrated the existence of a C4-hydroxylated vitamin D(2) metabolite in serum of rats treated with pharmacological doses of vitamin D(2). However, the biological significance and metabolic fate of this metabolite have not been described. To explore its potential biological activities, we therefore synthesized 1α,4α,25-trihydroxyvitamin D(3) and its diastereoisomer, 1α,4β,25-trihydroxyvitamin D(3), using Trost Pd-mediated coupling reaction, and studied their vitamin D receptor (VDR) binding affinity, osteocalcin promoter transactivation activity, and their further metabolism by human CYP24A1 as well as by human liver microsomal fraction based on CYP- and UDP-glucuronosyltransferases (UGTs)-reactions.

Effect of betaine on the hepatic damage from orotic acid-induced fatty liver development in rats

Betaine prevents hepatic damage caused by ethanol and carbone tetrachloride (CCl4) in rats. Present study was to investigate the effect of betaine on the hepatic microsomal triglyceride transfer protein (MTP) mRNA expression in orotic acid (OA)-induced fatty liver in rats. OA feeding was attributed to the significant increase in the hepatic levels of triglyceride and the serum levels of ALT and AST and resulted in typical histology of fatty liver contained numerous largely fat droplets. While concomitant supplementation of betaine to OA diet was slightly reduced the hepatic triglyceride concentrations and was significantly decreased ALT activity. Hepatic MTP mRNA expression by OA treatment increased by 14% despite triglyceride accumulation in the liver in OA treatment rats relative to rats fed a normal diet without OA supplemented, but MTP expression by simultaneous supplementation of OA and betaine was slightly decreased by 7.9% as compared to the OA-feeding rats. A significant elevation of TBARS contents in the liver homogenate, microsome, and mitochondrial fractions of the OA-feeding rats compared with the normal rats, however, these increases were significantly or slightly decreased by simultaneous addition of OA and betaine. The increases of hepatic OA and betaine levels in OA feeding rats was also found when compared to the normal rats, but these increases were significantly lowered in the concomitant supplementation OA and betaine. The content of Fe was significantly increased in the OA feeding rats, but this elevation showed significantly recovered as low as the normal level by concomitant with OA and betaine. Zinc content was also significantly decreased in the OA feeding rats compared with the normal rats, but this reduction was more significantly elevated by concomitant with OA and betaine. Hepatic glutathione content in the OA feeding rats was similar to that of the normal rats, but this content was slightly reduced without statistically significant differences. But, a significant elevation in the hepatic glutathione content was found in the simultaneously administration OA and betaine. The hepatocytes contained numerous largely fat droplets induced by OA administration and was slightly reduced by simultaneous supplementation of OA and betaine. Present study demonstrated that betaine has a weak preventive action on the OA-induced triglyceride accumulation.

Stereoselective liquid chromatographic determination of 1′‐oxobufuralol and 1′‐hydroxybufuralol in rat liver microsomal fraction using hollow‐fiber liquid‐phase microextraction for sample preparation

A three-phase hollow-fiber liquid-phase microextraction (HF-LPME) method for the stereoselective determination of bufuralol metabolites 1'-oxobufuralol (1'-Oxo-BF) and 1'-hydroxybufuralol (1'-OH-BF) in microsomal preparations is described for the first time. The HPLC analysis was carried out using a Chiralcel OD-H column with hexane/2-propanol/methanol (97.5:2.0:0.5, v/v/v) plus 0.5% diethylamine as the mobile phase, and UV detection at 248 and 273 nm. The HF-LPME optimized conditions involved: n-octanol as the organic solvent, 0.2 mol/L acetic acid as the acceptor phase, donor phase pH adjusted to 13, sample agitation at 1500 rpm and extraction for 30 min. By using this extraction procedure, the recovery rates were in the range of 63-69%. The method was linear over the concentration range of 100-5000 ng/mL for each enantiomer of 1'-Oxo-BF (r>0.9978) and of 100-2500 ng/mL for each stereoisomer of 1'-OH-BF (r>0.9957). The quantification limits were 100 ng/mL for all analytes. The validated method was used to assess the in vitro biotransformation of bufuralol using rat liver microsomal fraction that demonstrated predominant formation of (S)-1'-Oxo-BF and (R,R)-1'-OH-BF.

Significantly reduced cytochrome P450 3A4 expression and activity in liver from humans with diabetes mellitus

Patients with diabetes mellitus require pharmacotherapy with numerous medications. However, the effect of diabetes on drug biotransformation is not well understood. Our goal was to investigate the effect of diabetes on liver cytochrome P450 3As, the most abundant phase I drug-metabolizing enzymes in humans. Human liver microsomal fractions (HLMs) were prepared from diabetic (n= 12) and demographically matched nondiabetic (n= 12) donors, genotyped for CYP3A4*1B and CYP3A5*3 polymorphisms. Cytochrome P450 3A4, 3A5 and 2E1 mRNA expression, protein level and enzymatic activity were compared between the two groups. Midazolam 1'- or 4-hydroxylation and testosterone 6β-hydroxylation, catalyzed by P450 3A, were markedly reduced in diabetic HLMs, irrespective of genotype. Significantly lower P450 3A4 protein and comparable mRNA levels were observed in diabetic HLMs. In contrast, neither P450 3A5 protein level nor mRNA expression differed significantly between the two groups. Concurrently, we have observed increased P450 2E1 protein level and higher chlorzoxazone 6-hydroxylation activity in diabetic HLMs. These studies indicate that diabetes is associated with a significant decrease in hepatic P450 3A4 enzymatic activity and protein level. This finding could be clinically relevant for diabetic patients who have additional comorbidities and are receiving multiple medications. To further characterize the effect of diabetes on P450 3A4 activity, a well-controlled clinical study in diabetic patients is warranted.

The Imidazoacridinone Antitumor Drug, C-1311, Is Metabolized by Flavin Monooxygenases but Not by Cytochrome P450s

5-Diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) is an antitumor agent that is also active against autoimmune diseases. The intention of the present studies was to elucidate the role of selected liver enzymes in metabolism of C-1311 and the less active 8-methyl derivative, 5-diethylaminoethylamino-8-methoxyimidazoacridinone (C-1330). Compounds were incubated with rat liver microsomal fraction, with a set of 16 human liver protein samples, and with human recombinant isoenzymes of cytochrome P450, flavin monooxygenases (FMO), and UDP-glucuronosyltransferase (UGT). Our results showed that C-1311 and C-1330 were metabolized with human liver microsomal enzymes but not with any tested human recombinant cytochromes P450 (P450s). Two of these, CYP1A2 and CYP3A4, were inhibited by both compounds. In addition, results of C-1311 elimination from hepatic reductase-null mice, in which liver NADPH-P450 oxidoreductase has been deleted indicated that liver P450s were slightly engaged in drug transformation. In contrast, both compounds were good substrates for human recombinant FMO1 and FMO3 but not for FMO5. The product of FMO metabolism, P(FMO), which is identified as an N-oxide derivative, was identical to P3(R) of liver microsomes. P3(R) was observed even in the presence of the P450 inhibitor, 1-aminobenzotriazole, and it disappeared after heating. Therefore, FMO enzymes could be responsible for microsomal metabolism to P3(R) = P(FMO). Glucuronidation on the 8-hydroxyl group of C-1311 was observed with liver microsomes supported by UDP-glucuronic acid and with recombinant UGT1A1, but it was not the case with UGT2B7. Summing up, we showed that, whereas liver P450 isoenzymes were involved in the metabolism of C-1311 to a limited extent, FMO plays a significant role in the microsomal transformations of this compound, which is also a specific substrate of UGT1A1.

Aldehyde Reduction by Cytochrome P450

This protocol describes the procedure for measuring the relative rates of metabolism of the α,β-unsaturated aldehydes 9-anthracene aldehyde (9-AA) and 4-hydroxy-trans-2-nonenal (4-HNE). More specifically, these assays measure the aldehyde reduction reactions of cytochrome P450s (CYPs). They can be performed using liver microsomal or other tissue fractions, spherosome preparations of recombinant CYPs, or recombinant CYPs from other sources. The method for reduction of 9-AA (a model α,β-unsaturated aldehyde) by CYPs was adapted from an assay for 9-anthracene oxidation published by Marini et al. (2003). For reduction of the endogenous aldehyde 4-HNE, the substrate was incubated with CYP in the presence of oxygen and NADPH, and the metabolites were separated by HPLC, using an adaptation of the method by Srivastava et al. (2010). For both 9-AA and 4-HNE, the first step involves incubation of the substrate with the CYP in an appropriate medium. This is followed by quantification of metabolites through by spectrofluorometry (9-AA) or HPLC coupled with a radiometric assay (4-HNE). Metabolite identification can be achieved by HPLC GC/MS analysis. Inhibitors of cytochrome P450 can be utilized to show the role of the hemoprotein or other enzymes in these reduction reactions. The reduction of CYPs is not inhibited by either anaerobiosis or inclusion of CO in the gaseous phase of the reaction mixture. These characteristics are similar to those reported for some cytochrome P450-catalyzed azo reduction reactions.

Evidence for differences in regioselective and stereoselective glucuronidation of silybin diastereomers from milk thistle (Silybum marianum) by human UDP-glucuronosyltransferases

The flavonolignan silybin, the main component of silymarin, extract from the seeds of Silybum marianum, is used mostly as a hepatoprotectant. Silybin is almost 1:1 mixture of two diastereomers A and B. The individual UDP-glucuronosyltransferases (UGTs) contributing to the metabolism of silybin diastereomers have not been identified yet. In this study, the contribution of UGTs to silybin metabolism was examined.The potential silybin metabolites were formed in vitro by incubating silybin (i) with the human liver microsomal fraction, (ii) with human hepatocytes and finally (iii) with 12 recombinant UGTs (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17). High-performance liquid chromatographic (HPLC) techniques with UV detection and additionally MS detection were used for metabolite identification.Hepatocytes and microsomes formed silybin A-7-O-β-d-glucuronides, B-7-O-β-d-glucuronides, A-20-O-β-d-glucuronides and B-20-O-β-d-glucuronides. With recombinant UGTs, the major role of the UGT1A1, 1A3, 1A8 and 1A10 enzymes but also of the UGT1A6, 1A7, 1A9, 2B7 and 2B15 in the stereoselective reactions leading to the respective silybin glucuronides was confirmed. UGT1A4, UGT2B4 and UGT2B17 did not participate in silybin glucuronidation.The predominant formation of 7-O-β-d-glucuronides and the preferential glucuronidation of silybin B diastereomer in vitro by human UGTs were confirmed.

In vitro characterization of rosiglitazone metabolites and determination of the kinetic parameters employing rat liver microsomal fraction

Rosiglitazone (RSG), a thiazolidinedione antidiabetic drug, is metabolized by CYP450 enzymes into two main metabolites: N-desmethyl rosiglitazone (N-Dm-R) and ρ-hydroxy rosiglitazone (ρ-OH-R). In humans, CYP2C8 appears to have a major role in RSG metabolism. On the other hand, the in vitro metabolism of RSG in animals has not been described in literature yet. Based on these concerns, the kinetic metabolism study of RSG using rat liver microsomal fraction is described for the first time. Maximum velocity (V (max)) values of 87.29 and 51.09 nmol/min/mg protein were observed for N-Dm-R and ρ-OH-R, respectively. Michaelis-Menten constant (K(m)) values were of 58.12 and 78.52 μM for N-Dm-R and ρ-OH-R, respectively. Therefore, these results demonstrated that this in vitro metabolism model presents the capacity of forming higher levels of N-Dm-R than of ρ-OH-R, which also happens in humans. Three other metabolites were identified employing mass spectrometry detection under positive electrospray ionization: ortho-hydroxy-rosiglitazone (ο-OH-R) and two isomers of N-desmethyl hydroxy-rosiglitazone. These metabolites have also been observed in humans. The results observed in this study indicate that rats could be a satisfactory model for RSG metabolism.

The identification and analysis of the metabolic products of mephentermine.

Phentermine (Ib), N-hydroxymephentermine (Ic) and N-hydroxyphentermine (Id) were identified as metabolic products after in vitro incubation of mephentermine (Ia) with rabbit liver microsomal fractions. Compounds Ia, Ib and Ic were also identified as excretion products in the urine of a human subject given a single dose of mephentermine (Ia) sulphate. Derivatization with acetic anhydride, trifluoroacetic anhydride and the trimethylsilyl donor reagent N,O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) or hexamethyldisilazane (HMDS) were used for qualitative identification of the metabolic products Ib-Id by g.l.c.-mass spectrometry and for quantitative determination of Ia-Id after extraction from rabbit hepatic homogenates. The synthesis of N-hydroxymephentermine (Ic) and the properties of the metabolic products are reported.

N-ethyl-alpha-methyl-alpha-(m-trifluoromethylbenzyl)nitrone--the major in vitro metabolic product of fenfluramine in guinea-pig liver microsomal fractions.

N-ethyl-alpha-methyl-alpha-(m-trifluoromethylbenzyl)nitrone--the major in vitro metabolic product of fenfluramine in guinea-pig liver microsomal fractions.

Metabolic N- and alpha-C-oxidation of norephedrine by rabbit liver microsomal fractions and synthesis of the metabolic pdocuts.

Abstract The products of metabolism of norephedrine by 10 000 g fractions of rabbit liver microsomes have been shown to be norephedrine hydroxylamine, l-hydroxy-2-oxo-l-phenylpropane oxime, 1-hydroxy-2-oxo-1-phenylpropane, 1,2-dihydroxy-l-phenylpropane (erythro). N-Oxidation was at least as important as α-C-oxidation. The synthesis and properties of the metabolic products are described.

The microsomal N-oxidation of phentermine.

The microsomal N-oxidation of phentermine (Ia) to N-hydroxyphentermine (Ib) and to alpha,alpha-dimethyl-alpha-nitroso-beta-phenylethane (Ic was investigated. Maximum activities were obtained with microsomal (9000 g supernatant and microsomes) fractions of rabbit liver in the presence of an NADPH generating system. Incubation of Ia with hepatic washed microsomes from a phenobarbitone pretreated rabbit increased the formation of Ib and decreased that of Ic but the total amount of N-oxidized metabolites (i.e. Ib + Ic) was not affected. The ratio of the metabolically produced Ic to Ib but not the total amount of N-oxygenated metabolites varied greatly depending of the liver microsomal fractions used in the incubation mixtures of Ia; more Ib was produced from Ia using 9000 g supernatant and conversely, more Ic was formed using the washed microsomes of the same liver. The nitroso compound (Ic) was metabolically reduced to Ib and Ib to Ia by the hepatic 9000 g supernatant and soluble fraction; under the same conditions, washed microsomes had only limited reductive properties towards Ic and Ib. N-Hydroxyphentermine (Ib) was not metabolically oxidized to Ic when incubated with washed microsomes from rabbit liver. The use of known carbon-oxidation inhibitors showed that cytochrome P-450 is not involved in the incorporation of oxygen at the nitrogen centre of Ia. The metabolic characteristics and kinetic behavior of the microsomal N-oxidation of Ia supported a recently proposed mechanism explaining the independent formation of Ib and Ic from a common precursor resulting from metabolic N-oxidation of Ia.

Ibuprofen Metabolism in the Liver and Gill of Rainbow Trout, Oncorhynchus mykiss

The presence of pharmaceuticals in the environment has become an important topic of discussion with respect to pharmaceutical absorption, metabolism and elimination in fish. This study investigates the metabolism of ibuprofen by rainbow trout (Oncorhynchus mykiss). In vitro metabolic loss of parent compound was measured in gill and liver S9 and microsomal fractions. Metabolite analysis found 2-hydroxy-ibuprofen as the major metabolite in uninduced S9 fractions. Supplementing S9 fractions with UDPGA did not significantly enhance metabolism. Additionally, assays involving the induction and inhibition of specific CYP isozymes support CYP1A2 as a possible metabolic pathway in fish.

Hollow fiber-based liquid-phase microextraction (HF-LPME) of isradipine and its main metabolite followed by chiral HPLC analysis: application to an in vitro biotransformation study

An enantioselective liquid chromatographic method using two-phase hollow fiber liquid-phase microextraction (HF-LPME-HPLC) was developed for the determination of isradipine (ISR) enantiomers and its main metabolite (pyridine derivative of isradipine, PDI) in microsomal fractions isolated from rat liver. The analytes were extracted from 1 mL of microsomal medium using a two-phase HF-LPME procedure with hexyl acetate as the acceptor phase, 30 min of extraction, and sample agitation at 1,500 rpm. For the first time, ISR enantiomers and PDI were resolved. For this separation, a Chiralpak(®) AD column with hexane/2-propanol/ethanol (94:04:02, v/v/v) as the mobile phase at a flow rate of 1.5 mL min(-1) was used. The column was kept at 23 ± 2 °C. The drug and metabolite detection was performed at 325 nm and the internal standard oxybutynin was detected at 225 nm. The recoveries were 23% for PDI and 19% for each ISR enantiomer. The method presented quantification limits (LOQ) of 50 ng mL(-1) and was linear over the concentration range of 50-5,000 and 50-2,500 ng mL(-1) for PDI and each ISR enantiomer, respectively. The validated method was employed to an in vitro biotransformation study of ISR using rat liver microsomal fraction showing that (+)-(S)-ISR is preferentially biotransformed.

Genotoxicity of acrylamide in vitro: Acrylamide is not metabolically activated in standard in vitro systems

The recent finding that acrylamide (AA), a genotoxic rodent carcinogen, is formed during the frying or baking of a variety of foods raises human health concerns. AA is known to be metabolized by cytochrome P450 2E1 (CYP2E1) to glycidamide (GA), which is responsible for AA's in vivo genotoxicity and probable carcinogenicity. In in-vitro mammalian cell tests, however, AA genotoxicity is not enhanced by rat liver S9 or a human liver microsomal fraction. In an attempt to demonstrate the in vitro expression of AA genotoxicity, we employed Salmonella strains and human cell lines that overexpress human CYP2E1. In the umu test, however, AA was not genotoxic in the CYP2E1-expressing Salmonella strain or its parental strain. Moreover, a transgenic human lymphoblastoid cell line overexpressing CYP2E1 (h2E1v2) and its parental cell line (AHH-1) both showed equally weak cytotoxic and genotoxic responses to high (>1 mM) AA concentrations. The DNA adduct N7-GA-Gua, which is detected in liver following AA treatment in vivo, was not substantially formed in the in vitro system. These results indicate that AA was not metabolically activated to GA in vitro. Thus, AA is not relevantly genotoxic in vitro, although its in vivo genotoxicity was clearly demonstrated.

Anthracyclines and their metabolism in human liver microsomes and the participation of the new microsomal carbonyl reductase

Anthracyclines (ANTs) are widely used in the treatment of various forms of cancer. Although their usage contributes to an improvement in life expectancy, it is limited by severe adverse effects—acute and chronic cardiotoxicity. Several enzymes from both AKR and SDR superfamilies have been reported as participants in the reduction of ANTs. Nevertheless all of these are located in the cytosolic compartment. One microsomal reductase has been found to be involved in the metabolism of xenobiotics—11beta-HSD1, but no further information has been reported about its role in the metabolism of ANTs. The aim of this study is to bring new information about the biotransformation of doxorubicin (DOX), daunorubicin (DAUN) and idarubicin (IDA), not only in human liver microsomal fraction, but also by a novel human liver microsomal carbonyl reductase that has been purified by our group. The reduction of ANTs at C-13 position is regarded as the main pathway in the biotransformation of ANTs. However, our experiments with human liver microsomal fraction show different behaviour, especially when the concentration of ANTs in the incubation mixture is increased. Microsomal fraction was incubated with doxorubicin, daunorubicin and idarubicin. DOX was both reduced into doxorubicinol (DOXOL) and hydrolyzed into aglycone DOX and then subsequently reduced. The same behaviour was observed for the metabolism of DAUN and IDA. The activity of hydrolases definitely brings a new look to the entire metabolism of ANTs in microsomal fraction, as formed aglycones undergo reduction and compete for the binding site with the main ANTs. Moreover, as there are two competitive reducing reactions present for all three ANTs, kinetic values of direct reduction and the reduction of aglycone were calculated. These results were compared to previously published data for human liver cytosol. In addition, the participation of the newly determined human liver microsomal carbonyl reductase was studied. No reduction of DOX into DOXOL was detected. Nevertheless, the involvement in reduction of DAUN into DAUNOL as well as IDA into IDAOL was demonstrated. The kinetic values obtained were then compared with data which have already been reported for cytosolic ANTs reductases.

Characterization of nuciferine metabolism by P450 enzymes and uridine diphosphate glucuronosyltransferases in liver microsomes from humans and animals

to characterize the metabolism of nuciferine by P450 enzymes and uridine diphosphate glucuronosyltransferase (UGT) in liver microsomes from humans and several other animals including rats, mice, dogs, rabbits and monkeys. nuciferine was incubated with both human and animal liver microsomal fractions containing P450 or UGT reaction components. Ultra performance liquid chromatography coupled with mass spectrometry was used to separate and identify nuciferine metabolites. Chemical inhibition was used to identify the involved isozymes. Species difference of nuciferine metabolism in human and various animals were investigated in the liver microsomal incubation system. among the nuciferine metabolites detected and identified, seven were catalyzed by P450 and one by UGT. Ketoconazole inhibited the formation of M292, M294 and M312. Furafylline, 8-methoxypsoralen and quercetin inhibited the formation of M282. Hecogenin showed a significant inhibitory effect on nuciferine glucuronidation. While the P450-catalyzed metabolites showed no species differences, the glucuronidation product was only detected in microsomes from humans and rabbits. the isozymes UGT 1A4, CYP 3A4, 1A2, 2A6 and 2C8 participated in the hepatic metabolism of nuciferine. Based on the observed species-specific hepatic metabolism of nuciferine, rats, mice, dogs and even monkeys are not suitable models for the pharmacokinetics of nuciferine in humans.

Gas-phase photocatalytic degradation and detoxification of o-toluidine: Degradation mechanism and Salmonella mutagenicity assessment of mixed gaseous intermediates

The photocatalytic degradation of toluidine over titanium oxide (TiO 2) thin films under UV irradiation was investigated. The degradation efficiency of 98.7% was obtained for a toluidine concentration of about 4500 μg L −1 and illumination of 240 min. The degradation intermediates produced during photocatalytic oxidation were identified using Fourier transform-infrared spectrometry (FTIR) and gas chromatography–mass spectrometry (GC–MS). Only a small amount of intermediates, including phenol and toluene, were found in the gas phase. Many other trace amount intermediates, such as 2-hydroxybenzaldehyde, 2-nitrobenzaldehyde, 2-hydroxybenzenemethanol, 2-hydroxybenzoic acid, phenol etc., were detected on the TiO 2 surface. An Ames assay of the Salmonella typhimurium strains TA98 and TA100 was employed to evaluate the mutagenicity of toluidine and its gaseous photocatalytic degradation intermediates. With or without rat liver microsomal fraction (S9 mix) activation, neither toluidine nor its gaseous intermediates presented mutagenic activity against strains TA98 (±S9) and TA100 (−S9) at all tested doses. Toluidine, however, can induce a weak positive response to the TA100 strain with an S9 mix at doses as high as 4000 μg plate −1. An increase of revertants per plate was obtained after 30 min photocatalysis in the TA100 strain with S9 mix. As reaction time further increased, photocatalytic technology exhibited the ability to completely and efficiently detoxify toluidine. Both our chemical analysis and toxic evaluation indicate that all mutagenic intermediates in the gas can be completely eliminated within 240 min, which further suggests that photocatalytic technology is an effective approach for degrading aromatic amines.