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. 2016 Feb 7:38:1.
doi: 10.1186/s41021-016-0028-x. eCollection 2016.

Modified Ames test using a strain expressing human sulfotransferase 1C2 to assess the mutagenicity of methyleugenol

Hiroshi Honda  1 , Kazuyuki Minegawa  2 , Yurika Fujita  1 , Noriko Yamaguchi  2 , Yoshihiro Oguma  2 , Hansruedi Glatt  3 , Naohiro Nishiyama  1 , Toshio Kasamatsu  1
Affiliations

Modified Ames test using a strain expressing human sulfotransferase 1C2 to assess the mutagenicity of methyleugenol

Hiroshi Honda et al. Genes Environ. .

Abstract

Introduction: Several alkenylbenzenes, including methyleugenol (ME), are present in a wide range of botanicals and exhibit carcinogenic and mutagenic properties. Negative results are generally obtained for alkenylbenzenes in standard in vitro genotoxicity tests, including the Ames test. A lack of mutagenicity observed in such tests is thought to result from impaired metabolic activation of alkenylbenzenes via hydroxylation, with subsequent sulfoconjugation to its ultimate mutagenic or carcinogenic form. Although recent studies have reported the mutagenicity of hydroxylated ME metabolites in the Ames test using modified TA100 strains expressing human sulfotransferases (SULTs), to our knowledge, the detection of ME mutagenicity has not yet been reported.

Findings: Using strain TA100-hSULT1C2, which expresses human SULT1C2, we optimized the protein content of S9 Mix and the pre-incubation time required to promote metabolic activation in the Ames test. This procedure enabled us to obtain a positive response with ME.

Conclusions: We established Ames-test conditions enabling the detection of ME-induced mutagenicity, using a strain expressing human SULT1C2 in the presence of induced-rat S9 Mix. This simple approach will help assess the mutagenicity of other alkenylbenzenes and related chemicals.

Keywords: Alkenylbenzene; Ames test; Methyleugenol; S9; Sulfotransferase.

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Figures

Fig. 1
Fig. 1
Metabolic-activation pathway of methyleugenol (ME) leading to the production of mutagenic metabolites. ME is metabolized by cytochrome P450 (CYP) to 1’-HME. Subsequent sulfoconjugation of 1’-HME by sulfotransferase (SULT) leads to the production of highly reactive electrophiles that can form DNA adducts
Fig. 2
Fig. 2
Optimizing the protein content in the S9 Mix. Based on the results of the preliminary investigation, the total protein contents in the S9 Mix were set at 0, 0.3, 0.6, 1.2, or 2.4 mg/plate. The pre-incubation time was set at 1 h. The plots indicate the ratio of the average number of revertant colonies observed (treated: solvent control) in two plates and the number of spontaneous revertants. The number of spontaneous colonies per plate ranged from 108 to 151, varying slightly in response to the different protein levels used
Fig. 3
Fig. 3
Optimization of the pre-incubation time. The pre-incubation time was set at 0 min, 20 min, 1 h, or 2 h. The protein content in the S9 Mix was set at 1.2 mg/plate. The plots shown indicate the ratio of the average number of revertant colonies observed (treated: solvent control) in two plates and the number of spontaneous revertants. The number of spontaneous colonies per plate ranged from 100 to 133, varying slightly in response to the different pre-incubation times used
Fig. 4
Fig. 4
Results obtained under optimized conditions. The total protein content in the S9 Mix was set at 1.2 mg/plate (a common concentration for Ames tests), and the length of the pre-incubation period was 2 h for the main tests. The plots shown indicate the relative ratio of the average number of revertant colonies observed (treated: solvent control) in two plates and the number of spontaneous revertants. The number of spontaneous colonies per plate ranged from 96 to 128, varying slightly in the presence and absence of the S9 Mix in response to the different pre-incubation times used
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