Int J Pharm Pharm Sci, Vol 10, Issue 1, 19-22Original Article


MUTATIONAL SPECIFICITY OF 2, 6-DIMETHYLANILINE WITH IN VITRO HUMAN S9 METABOLIC ACTIVATION IN THE GPT GENE OF AS52 CELLS

SEO HYUN MOON1, MIN-YOUNG KIM2*

1Department of Forensic DNA, National Forensic Service, Wonju, Gangwon-do, Republic of Korea, 2Toxicology Laboratory, Faculty of Biotechnology (Biomaterials), College of Applied Life Science, SARI, Jeju National University, Jeju, Republic of Korea
Email: jeffmkim@jejunu.ac.kr

Received: 14 Oct 2017 Revised and Accepted: 22 Nov 2017


ABSTRACT

Objective: The purpose of the current work was to characterize mechanisms of cytotoxicity and mutagenesis of a potential human bladder carcinogen 2,6-dimethylaniline (2,6-DMA).

Methods: Chinese hamster ovary (CHO) AS52 cells were exposed to either human S9 activated 2,6-DMA for 6 h or its N-hydroxylamine and aminophenol metabolites for 1 h in serum-free medium. Cell survival determined by trypan blue exclusion 24 h after treatment, and 6-thioguanine-resistant mutants at the xanthine-guanine phosphoribosyltransferase (gpt) gene locus were assessed with doses of which relative survival is 30% or more. Nested PCR-based deletion analysis was also performed.

Results: AS52 cells exhibited a dose-dependent increase in cytotoxicity and mutant fraction upon treatment of 2,6-DMA and its metabolites, but showing considerable variation in potency with aminophenol metabolites having the highest potency and parent compound at least; at the highest 2,6-dimethyaminophenol dose (10 μM), the mutant fraction in AS52 cells was 8 fold (13.2×10-5) greater than the spontaneous fraction of 1.62×10-5. Total deletion of the gpt gene sequences was found in 1 (4%) of spontaneous and 2 (6%) of the 6-thioguanine mutants generated by N-hydroxy-2,6-DMA.

Conclusion: These findings indicate the mutagenicity of 2,6-DMA at the gpt gene, which is mediated through hydroxylamine and aminophenol metabolites, and contribute to the elucidation of mechanisms through which 2,6-DMA may exert its effects in vivo.

Keywords: 2,6-Dimethylaniline, Metabolic activation, Genotoxicity, AS52/gpt assay


INTRODUCTION

2,6-Dimethylaniline (2,6-DMA), a well-known alkyl anilines, is used as a chemical intermediate in the manufacture of pesticides, dyestuffs, antioxidants, pharmaceuticals, synthetic resins, fragrances and other products [1, 2]. Exposure to alkylaniline has been well documented in cigarette smokers [3], but also may occur from non-smoking related sources [4]. Smoking is associated with increased rates of bladder cancer [5]. Alkylamines are believed to be the constituents of tobacco smoke that lead to the development of bladder cancer [6, 7]. Several members of alkylanilines including 4-aminobiphenyl (4-ABP), 2-naphthylamine and benzidine, which are extensive, have been classified and regulated as human bladder carcinogens [8]. Other alkylaniline, including 2,6-DMA, have been implicated as possible human bladder carcinogens [6, 9-12]. Genotoxicity of alkylanilines has received very limited investigation to date, and the fragmented available database supports only limited conclusions. Alkylaniline is metabolized to reactive alkyl-hydroxylamine which can undergo further bioactivation and conjugation reactions [13-15].

These reactions include those catalyzed by cytochrome P450-dependent mono­oxygenases and other enzymes; the free reactive hydroxylamine may be regenerated from the conjugated form in an acidic environment [13-15]. Possible metabolic pathways leading to the formation of genotoxic DMA damage products of 2,6-DMA is shown in fig. 1. Recent results described here indicate that alkylanilines can be potent genotoxins for cultured mammalian cells when activated by exogenous or endogenous phase I and phase II xenobiotic-metabolizing enzymes [6, 7, 10-12, 15]. However, there is little information is available about mutagenicity of 2,6-DMA and a detailed metabolic profile of 2,6-DMA. Moreover, it is not clear whether 2,6-DMA can induce the deletions and other gross structural changes in other types of cells. Therefore, there is of considerable interest to examine the toxic effects of 2,6-DMA in the reportedly more sensitive in vitro system for mutation assay.

Fig. 1: Schematic representation of the presumed metabolic pathway for 2,6-DMA, Information was taken from Gan et al. (2001)

For this reason, we have introduced Chinese hamster ovary (CHO) AS52 cell line that carries a single copy of E. coli xanthine-guanine phosphoribosyl transferase (gpt) gene functionally expressed using SV40 early promoter and stably integrated into the AS52 cell genome [16]. AS52 cells were constructed by transfecting the plasmid vector pSVgpt into a normal X-linked mammalian HPRT-deficient CHO cells. The AS52 cell line has been demonstrated to be useful with regard to the detection of both deletion and point mutations at gpt, and the low spontaneous mutation fraction (MF) and the distinct advantage of carrying a small easily manipulated mutational target make the AS52 cell line particularly suited to quantitative and molecular mutagenesis studies. The gpt structural gene is 456 base pairs (bp) with no introns versus a structural gene size of 654 bp for HPRT, which with introns comprises a genomic HPRT locus of approximately 35 kb [17]. Thus, AS52 cells are sensitive to induced mutagenesis by a variety of clastogens and radiomimetic agents that are often classified as nonmutagens in other assays [17]. The purpose of the present study was to characterize the mutational profile and mechanism induced by 2,6-DMA in the gpt gene of AS52 cells. We have tested the hypothesis that toxic effects of 2,6-DMA are mediated through its hydroxylamine and/or aminophenol metabolite.

MATERIALS AND METHODS

Chemicals and reagents

All cell culture reagents were purchased from Lonza (Walkersville, MD). 2,6-dimethylaniline (2,6-DMA), 6-thioguanine (6-TG), dimethyl sulfoxide (DMSO), ethyl methansulfonate (EMS) and DL-isocitric acid were purchased from Sigma Chemical Co., St Louis, MO. 3,5-dimethylaniline (3,5-DMA) and NADP were obtained from Acros Organics (Geel, Belgium) and Boehringer Mannheim (Indianapolis, IN), respectively.

Cell cultures

Chinese hamster ovary AS52 cells, kindly provided by Dr. Gerald N. Wogan (Massachusetts Institute of Technology, Cambridge, MA, USA), were cultured in Ham’s F-12 medium supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin and 10% heat-inactivated fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) in a humidified atmosphere with 5% CO2 at 37 °C. Cells were cleansed of preexisting gpt mutants by culturing in MPA medium (10 μg/ml MPA, 250 µg/ml xanthine, 22 µg/ml adenine, 11 µg/ml thymidine and 1.2 µg/ml aminopterin) for 7 d and transferred for 3 d to medium enriched with xanthine (11.5 µg/ml), adenine (3 µg/ml) and thymidine (1.2 µg/ml) for recovery.

2,6-DMA treatment

Cells were placed in 6 well plates at a density of 0.5 × 106 cells per well the day before treatment. Cultures were, just prior to treatment, washed with PBS and refed with serum-free medium containing human liver S9 mix (Moltox Inc., Boone, NC). Approximately 440 µg S9 protein (16 µl fraction) and 65 µl sterile-filtered core mixture contained 25 mg/ml NADP and 45 mg/ml DL-isocitric acid were added per ml of serum-free medium. Cells were exposed to 25, 50, 100, 250, 500 and 1000 µM of 2,6-DMA dissolved in DMSO for 5 h at 37 °C, after which test compounds containing medium was removed and replaced with fresh medium supplemented with 10% FBS. The final concentration of DMSO to which cells µl were exposed was less than 0.1%.

Metabolites treatment

N-hydroxy and aminophenol metabolites of 2,6-DMA were synthesized as described previously [12]. The cells were seeded at 1 × 106 and incubated overnight with the regular medium. Then medium changed to one without serum and cells were treated in triplicate with 5, 10, 25, 50, 100 and 250 µM of N-hydroxy or aminophenol metabolites. After 1 h treatment, the culture was washed with PBS, amended with Ham’s F-12 medium containing 10% FBS, and incubated.

Measurement of survival and gpt mutations

Cytotoxicity was determined by trypan blue exclusion assay 24 h after treatment, and the cells were maintained for full expression of the mutant phenotype. Seven days after treatment, 5 × 105 cells from each group was placed in 100 ml of selection medium containing 10 μM 6-TG and plated at 50000 cells/10 ml/100 mm dish (10 dishes per group) for determination of mutagenicity. Simultaneously with the 6-TG selection, cells were seeded for determination of plating efficiency (200 cells/10 ml/100 mm dish; 5 dishes per group). Colonies were scored after incubation for 14 d, and the mutant fraction was calculated as the number of 6-TG mutants. The mutation fraction (MF) was calculated by dividing the number of mutants by the number of cells seeded and the plating efficiency. In one of the experiments, exposure to 8 mmol ethyl methanesulfonate (EMS) for 2 h was used as a positive control for mutation induction. Identified single gpt mutant colony was then grown to approximately 2 × 106 mutant cells for molecular analysis.

DNA isolation and PCR deletion screening

Identified single gpt mutant colony was transferred to 24 well plates for propagating mutant cells. Approximately 2 × 106 mutant cells were collected for molecular analysis. Genomic DNA extracted from each mutant by using a GenEluteTM mammalian genomic DNA miniprep kit (Sigma). Amplification of the gDNA was performed in two rounds of nested PCR in PTC-200 DNA Engine Thermal Cycler (Bio-Rad, Hercules, CA). A 1 µg of template DNA was transferred in to the first round mix of 10 μl 10×PCR buffer, 2 μl dNTP mix, 0.5 μl taq polymerase, 73.5 μl HPLC water, 0.2 μl each 25 mmol forward (bases-199 to-181; 5'-AAGCTTGGACACAAGACAG-3') and reverse (bases 520 to 540; 5'-CCAGAATACTTACTGGAAAC-3') primer and amplified with a PCR profile of 94 °C: 1 min, 30 cycles of 94 °C: 1 min, 47 °C: 1 min, 72 °C: 1 min and a final extension of 72 °C for 7 min. The product from this reaction was filtered using Centricon 50 concentrator (Amicon, Beverly, MA) and resuspended in 100 μl sterile water to avoid unspecific binding with remaining primers, and 10 μl aliquot was used as template in the second round of PCR using nested primers (bases-23 to-4; 5'-ATAAACAGG-CTGGGACACTT-3' and bases 460 to 470; 5'-AGTGCCAGGCG-TTGAAAAGA-3'). The PCR conditions were the same in the second round as those in the first round reaction, except annealing temperature (52 °C). A quantity of gpt gene amplification was analyzed by electrophoresis on 1% agarose gels stained with ethidium bromide.

RESULTS AND DISCUSSION

Survival and mutation fractions

Alkylaniline toxicity is characterized by methemoglobin formation and by hyperplasia and siderosis of the spleen [18]. The compound is nonmutagenic in the standard Salmonella typhimurium/microsome test [19]. It is positive, however, in inducing DNA damage in the liver and kidney of rats and in inducing sister chromatid exchanges in vivo in mice [20]. Alkylaniline, at a high concentration, induces minimal increases in sister chromatid exchanges in vitro [21] and forms DNA adducts [6, 11]. Mutagenic activity of a chemical is a function of both the capacity to adduct DNA and the capacity of the cell to respond to the damage. DNA adducts formed from distinct alkyl anilines have already been shown to be differentially mutagenic [22].

In the present study, all test compounds reduced the cell survival whereas they increased the proportion of cells carrying the mutated gpt gene in a dose-dependent manner (fig. 2 and 3). The survival of the test cell lines following 2,6-dimethyaminophenol (2,6-DMAP) treatment was lower than that observed in the corresponding N-hydroxy metabolite and parent compound; for instance, cell viability was 3% in AS52 cells 24 h after a dose of 50 μM 2,6-DMAP, compared to 69 and 85% after treatment with N-hydroxy-2,6-DMA (N-OH-2,6-DMA) and 2,6-DMA, respectively (fig. 2).

Fig. 2: Dose-dependent of cell survival after treatment with 2,6-DMA, and its N-hydroxy and aminophenol metabolites in AS52 cells, Survival was determined by trypan blue assay 24 h after treatment, Data represent mean±SD for three measurements

Previous studies showed that if the relative survive rate is 30% or more after chemical exposure, there will be sufficient live cells to determine mutation responses [23-24].

Fig. 3: Induced Mutation fraction in AS52 cells treated with 2,6-DMA, and its N-hydroxy and aminophenol metabolites, Data represent mean±SD for three measurements

Thus, based on the results of cytotoxicity experiment, three cultures with those concentration ranges for 2,6-DMA (25-500 μM), N-OH-2,6-DMA (5-100 μM) and 2,6-DMAP (5 and 10 μM) were used to determine mutagenic responses (fig. 3). When cells were treated with 100 μM of 2,6-DMA and N-OH-2,6-DMA, mutation fraction (MF) was 5.01 × 10-5 and 7.17 × 10-5, 3-and 4.5-fold the spontaneous MF (1.61 × 10-5), respectively. On the other hand, 5 μM 2,6-DMAP, which is a 20 times lower concentration, enhanced the MF from 1.61 × 10-5 to 6.48 × 10-5 (fig. 3). By comparison, in EMS-treated positive controls, MF at the gpt locus was 72.7 × 10-5.

Our results correlated well with that obtained by previous studies showing both phases I and phase II metabolism are required for genotoxic specific-activation of 2,6-DMA. Nohmi et al. reported the presence of the mutagenic N-hydroxylated metabolite of 2,4-DMA in an in vitro rat liver microsomal system (S9) in which the yield of the mutagenic metabolite increased with increasing S9 content [25]. Beland et al. reported the mutagenicity of the N-­hydroxylated metabolite of 2,6-DMA towards Salmonella typhimurium (TA100) [26].

Deletion screening of AS52 gpt mutants

Thirty-five independent 500 μM 2,6-DMA and 100 μM N-OH-2,6-DMA, 34 independent 10 μM 2,6-DMAP and 24 independent independent spontaneous 6-TG-resistant mutants in AS52 cells were isolated and analyzed by the nested PCR, to test for the presence or absence of the gpt structural gene (0.5 kb PCR product) and the closely linked rearrangement product of this gene (0.8 kb PCR product). Those 6-TG-resistant mutants with both PCR sequences intact are mostly likely to carry point mutations in the gpt structural gene. Loss or alteration of one or both bands suggests more complex chromosomal deletions [27] and/or rearrangements involving gpt [28-29].

Table 1: PCR analysis of gpt in AS52 Cells after treatment with 2,6-DMA, and its metabolites

Number of mutants (%)
Spontaneous 2,6-DMA N-OH-2,6-DMA 2,6-DMAP
No observable alterationa 23 (96%) 35 (100%) 33 (94%) 34 (100%)
Large deletions/rearrangementsb 1 (4%) 0 (0%) 2 (6%) 0 (0%)
Total of mutants analyzed 24 (100%) 35 (100%) 35 (100%) 34 (100%)

aNo alterations were observable in the migration of the primary PCR products of the structural gpt gene on an agarose gel as compared to the AS52 wild-type control, bLarge deletions or rearrangements resulting in the absence of PCR amplified gpt sequences [Complete deletions (0.5+0.8 kb) and deletions of the 0.5 kb with normal 0.8 kb product].

Screening results of the gpt gene deletions showed that the percentage of deletion mutations in the spontaneous gpt mutants was 4% (1 out of 24): that of the 100 μM N-OH-2,6-DMA-enhanced mutants was 6% (2 out of 35) (table 2 and fig. 4). The mechanisms for the gene deletion are complicated and may involve different pathways such as DNA strand breakage, error in DNA damage repair, and other kinds of DNA damages [30]. Deletion of the gpt gene in N-OH-2,6-DMA-enhanced mutants may be results from DNA damage caused by high cytotoxicity of the chemical.

Fig. 4: Nested PCR amplication of the gpt gene in AS52 cells, two prominent bands at 0.5 (gpt structural gene) and 0.8 kb (rearranged pSVgpt gene) are observed in wild-type AS52 cells (WT), Mutants generated by N-OH-2,6-DMA, rearranged pSVgpt band are present, but the PCR product for the structural gene is missing; 1kb ladders are shown in left lane

CONCLUSION

The results reveal that 2,6-DMA is mutagenic and genotoxic in gpt gene of AS52 cells when activated by phase I and phase II metabolism, in which the aminophenol metabolites were considerably more potent than the corresponding N-hydroxylamines. Further study is also needed to investigate the mechanism of getnotoxic activity of 2,6-DMA.

ACKNOWLEDGEMENT

This work was supported by Basic Science Research Program (2017R1D1A1B03028849, 2016R1A6A1A03012862 and 2014R1A1-A2056292) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology, Republic of Korea.

AUTHORS CONTRIBUTION

All authors had equally contributed the research work

CONFLICT OF INTERESTS

We declare that there is no conflict of interest

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About this article

Title

MUTATIONAL SPECIFICITY OF 2, 6-DIMETHYLANILINE WITH IN VITRO HUMAN S9 METABOLIC ACTIVATION IN THE GPT GENE OF AS52 CELLS

Keywords

2,6-Dimethylaniline, Metabolic activation, Genotoxicity, AS52/gpt assay

DOI

10.22159/ijpps.2018v10i1.23133

Date

01-01-2018

Additional Links

Manuscript Submission

Journal

International Journal of Pharmacy and Pharmaceutical Sciences
Vol 10, Issue 1, 2018 Page: 19-22

Online ISSN

0975-1491

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Authors & Affiliations

Seo Hyun Moon
Department of Forensic DNA, National Forensic Service, Wonju, Gangwon-do, Republic of Korea

Min Young Kim
Toxicology Laboratory, Faculty of Biotechnology (Biomaterials), College of Applied Life Science, SARI, Jeju National University, Jeju, Republic of Korea


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