Role of cytochrome P450 1A2 and N-acetyltransferase 2 in 2, 6-dimethylaniline induced genotoxicity

Abstract The purpose of the current work was to assess a possible role of cytochrome P450 1A2 (CYP1A2) and N-acetyltransferase 2 (NAT2) in the metabolic activation of 2,6-dimethylaniline (2,6-DMA) and also clarify the function of DNA repair in affecting the ultimate mutagenic potency. Two cell lines, nucleotide excision repair (NER)-deficient 5P3NAT2 and proficient 5P3NAT2R9 both expressing CYP1A2 and NAT2, were treated with 2,6-DMA for 48 h or its metabolites for 1 h. Cell survival determined by trypan blue exclusion and MTT assays, and 8-azaadenine-resistant mutants at the adenine phosphoribosyltransferase (aprt) gene locus were evaluated. 5P3NAT2 and 5P3NAT2R9 cells treated with 2,6-DMA and its metabolites showed a dose-dependent increase in cytotoxicity and mutant fraction; N-OH-2,6-DMA and 2,6-DMAP in serum-free α-minimal essential medium (MEM) are more potent than 2,6-DMA in complete MEM. 5P3NAT2 cells was more sensitive to the cytotoxic and mutagenic action than 5P3NAT2R9 cells. H2DCFH-DA assay showed dose-dependent ROS production under 2,6- DMAP treatment. These findings indicate that the genotoxic effects of 2,6-DMA are mediated by CYP1A2 activation via N-hydroxylation and the subsequent esterification by the phase II conjugation enzyme NAT2, and through the generation of ROS by hydroxylamine and/or aminophenol metabolites. NER status is also an important contributor

2,6-and 3,5-dimethylaniline-induced mutagenesis in Chinese hamster ovary cells expressing human cytochrome P450 1A2 and sulfotransferase

Aim: The aim of this study was to test the hypothesis that human cytochrome P450 1A2 (CYP1A2) and sulfotransferase (SULT) contribute to the phase I and II bioactivation of 2,6-dimethylaniline (2,6-DMA) and 3,5-dimethylaniline (3,5-DMA) in affecting the incidence of genotoxicity.Methodology: 5P3H1 cells carrying cytochrome P450 1A2 (CYP1A2) and SULT cells were treated with various concentrations of 2,6-and 3,5-DMA for 48 hr or their N-hydroxyl and aminophenol metabolites for 1 hr in the absence or presence of 2,6-Dichloro-4-nitrophenol (DCNP). Cell lethality was assayed by trypan blue exclusion and induced mutagenesis of adenine phosphoribosyl transferase (aprt) gene was also evaluated. Results: A significant dose-dependent increase in cytotoxicity and mutant fraction was observed after treatment with 2,6- and 3,5-DMA, and their metabolites; N-hydroxy and aminophenol metabolites are more potent than the parent compounds. Addition of sulfotransferase inhibitor DCNP decreased the cytotoxic and mutagenic effects of 2,6- and 3,5-DMA, and their metabolites in a dose-dependent manner. Interpretation: This research indicate that 2,6 and 3,5-DMA are mutagenic, and their toxicity in model systems depend on metabolic activation. This activation is mediated by CYP1A2 and SULT enzymes

The role of nucleotide excision repair on 2,6- and 3,5-dimethylaniline-induced genotoxicity

Aim: To examine the possible role of nucleotide excision repair (NER) in affecting the ultimate mutagenic potency of 2,6- and 3,5-dimethylaniline (DMA) and their metabolites.Methodology: Two cell lines, nucleotide excision repair (NER)-proficient AA8 and deficient UV5 cells were treated with 50, 100, 250, 500 and 1000 μM of 2,6- and 3,5-DMA for 48 hr or their N-hydroxyl and aminophenol metabolites for 1 hr. Cell survival was determined by trypan blue exclusion assay, and 8-azaadenine-resistant mutants at adenine phosphoribosyltransferase (aprt) gene locus were evaluated.Results: A dose-dependent increase in cytotoxicity and mutant fraction was observed in AA8 and UV5 cells, treated with 2,6- and 3,5-DMA and their metabolites, but showed considerable variation in potency; N-hydroxyl and aminophenol metabolites of 2,6- and 3,5-DMA in serum-free α-minimal essential medium (MEM) having the highest potency, and 2,6- and 3,5-DMA in regular MEM at least. Repair-deficient UV5 cells were more sensitive to cytotoxic and mutagenic action than repair-proficient AA8 cells. Interpretation: These findings suggest that 2,6- and 3,5-DMA-induced DNA damage response may trigger cytotoxicity and mutagenicity when not completely repaired

Simultaneous detection of 6 isomers of dimethylaniline in workplace air by gas chromatography / 中国职业医学

OBJECTIVE: To establish a method for simultaneous detection of 6 isomers of dimethylaniline(DMA) in workplace air by gas chromatography(GC). METHODS: The vapor and aerosol DMA in workplace air were collected by GDH-1 air sampling tube, and desorbed and eluted with absolute ethanol, then separated by a special capillary column for amines analysis, and finally detected by GC-flame ionization detector. RESULTS: The quantitative detection range of 6 isomers of DMA was 0.26-226.66 mg/L, with all the correlation coefficients greater than 0.999 00. The minimum detectable concentration was 0.01-0.02 mg/m~3, and the minimum quantification concentration was 0.04-0.05 mg/m~(3 )(15.00 L sample, 2.00 mL sample solution). The average desorption and elution efficiency was 92.48%-104.60%. The within-run relative standard deviations(RSD) was 0.31%-4.51%, and the between-run RSD was 1.23%-6.03%. The samples can be sealed and stored for more than 19 days at room temperature. CONCLUSION: The method is suitable for simultaneous detection of 6 isomers of DMA in workplace air.

Determination of N,N-Dimethylaniline in Quetiapine Fumarate by LC-MS / 医药导报

Objective To establish a LC-MS analytical method for determination of N,N-dimethylaniline which is genotoxic impurity in quetiapine fumarate.Methods The method was achieved by an Waters ACQUITY UPLC CSHTM PhenylHexyl(2.1 mm× 100 mm,1.7 μm) utilizing a mobile phase of buffer-methanol(900∶ 100) (A)-acetonitrile(B) with gradient elution at the flow rate of 0.4 mL·min-1.The temperature of column was set at 50 ℃;The DIONEX Ultimate 3000 HPLC-AB Science 4000 QTrap Tripling Four bar LC-MS to detect N,N-Dimethylaniline (ESI source,in MRM positive mode).Results Standard curve was linear in the range of 0.4-8.0 ng(r =0.999 3);The limit of detection was 0.2 ng;The limit of quantification of N,N-dimethylaniline was 0.4 ng,respectively.The average recovery of N,N-dimethylaniline was 103.3 ％;RSD was 4.3％ (n =9),respectively.Conclusion The method is convenient and sensitive for the determination of N,N-dimethylaniline in quetiapine fumarate.