A study for the genotoxicity assessment of substances containing probiotic candidates in the in vitro TK6 cell micronucleus test: Influence of low pH conditions and antibiotic supplementation on the test results.

IF 2.7 4区 医学 Q2 GENETICS & HEREDITY
Yohei Fujiishi, Wakako Ohyama, Emiko Okada
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引用次数: 0

Abstract

Background: When assessing the genotoxicity of substances containing probiotic candidates, such as lactic acid-producing bacteria, using the in vitro micronucleus test (MNT), bacterial growth in the test medium may reduce the pH of the medium. The low medium pH is known to induce cytotoxicity and false-positive results. In the TK6 cell system, it is difficult to completely remove the bacteria from the medium by washing post-treatment, leading to bacterial growth during the recovery period in the short-term treatment. In the present study, the low pH range yielding false positives in the TK6 cell MNT was investigated using media supplemented with acetic, lactic, or formic acids, which are non-genotoxic bacterial metabolites. Additionally, to suppress the bacterial growth during the recovery period using antibiotics, i.e., penicillin/streptomycin (P/S), gentamicin sulfate (GM), and amphotericin B (AP), the maximum applicable concentrations of them that did not affect TK6 cell growth or micronucleus induction were determined. Then, we conducted an MNT using a substance containing live lactic acid-producing bacteria to verify the effectiveness of the antibiotics.

Results: Acetic, lactic, and formic acids induced micronuclei in TK6 cells (false positive) at an initial pH of ≤ 6.2 and ≤ 6.0 in 3 h treatment with and without S9 mix, respectively, and of ≤ 6.7 in the continuous treatment. Media supplemented with P/S, GM, and AP did not affect TK6 cell growth or micronucleated cell frequencies in the negative and positive controls ≤ 400 unit/mL-400 µg/mL, ≤ 250, and ≤ 20 µg/mL, respectively. In an MNT with fermented milk containing live lactic acid-producing bacteria, supplementation with P/S or GM to media for the recovery cultures suppressed the bacterial growth, decreasing pH, and cytotoxicity.

Conclusion: This study revealed the low pH ranges yielding false positives in the TK6 cell MNT under short-term and continuous treatment conditions. These values will serve as references for interpreting the biological relevance of results. Under short-term treatment, optimal antibiotic supplementation in recovery cultures suppressed bacterial growth in the test substance and prevented the decrease in pH that could yield false positives. This approach might be useful for evaluating the genotoxicity of test substances containing probiotic candidates using the MNT.

体外TK6细胞微核试验中含有益生菌候选物质的遗传毒性评估研究:低pH条件和补充抗生素对试验结果的影响
背景:当使用体外微核试验(MNT)评估含有益生菌候选物质(如乳酸菌)的遗传毒性时,细菌在试验培养基中的生长可能会降低培养基的pH值。已知培养基的低pH值可诱导细胞毒性和假阳性结果。在TK6细胞体系中,处理后的冲洗很难完全去除培养基中的细菌,导致短期处理的恢复期细菌生长。在本研究中,使用添加乙酸、乳酸或甲酸的培养基研究了TK6细胞MNT中产生假阳性的低pH范围。乙酸、乳酸或甲酸是无基因毒性的细菌代谢物。此外,为了抑制恢复期细菌的生长,使用抗生素,即青霉素/链霉素(P/S)、硫酸庆大霉素(GM)和两性霉素B (AP),确定不影响TK6细胞生长和微核诱导的最大适用浓度。然后,我们使用含有活乳酸菌的物质进行了MNT,以验证抗生素的有效性。结果:醋酸、乳酸和甲酸分别在初始pH≤6.2和≤6.0时诱导TK6细胞微核(假阳性),加S9和不加S9混合物处理3 h,初始pH≤6.7,连续处理≤6.7。在≤400 unit/mL、≤250µg/mL和≤20µg/mL的阴性对照和阳性对照中,培养基中添加P/S、GM和AP对TK6细胞生长和微核细胞频率均无影响。在含有活乳酸菌的发酵乳的MNT中,在培养基中添加P/S或GM可抑制细菌生长,降低pH值和细胞毒性。结论:本研究揭示了在短期和持续治疗条件下,低pH范围的TK6细胞MNT产生假阳性。这些值将作为解释结果的生物学相关性的参考。在短期治疗下,在恢复培养物中补充最佳抗生素可抑制测试物质中的细菌生长,并防止可能产生假阳性的pH值下降。这种方法可能有助于使用MNT评估含有益生菌候选物的测试物质的遗传毒性。
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来源期刊
Genes and Environment
Genes and Environment Biochemistry, Genetics and Molecular Biology-Genetics
CiteScore
4.00
自引率
0.00%
发文量
24
审稿时长
27 weeks
期刊介绍: Genes and Environment is an open access, peer-reviewed journal that aims to accelerate communications among global scientists working in the field of genes and environment. The journal publishes articles across a broad range of topics including environmental mutagenesis and carcinogenesis, environmental genomics and epigenetics, molecular epidemiology, genetic toxicology and regulatory sciences. Topics published in the journal include, but are not limited to, mutagenesis and anti-mutagenesis in bacteria; genotoxicity in mammalian somatic cells; genotoxicity in germ cells; replication and repair; DNA damage; metabolic activation and inactivation; water and air pollution; ROS, NO and photoactivation; pharmaceuticals and anticancer agents; radiation; endocrine disrupters; indirect mutagenesis; threshold; new techniques for environmental mutagenesis studies; DNA methylation (enzymatic); structure activity relationship; chemoprevention of cancer; regulatory science. Genetic toxicology including risk evaluation for human health, validation studies on testing methods and subjects of guidelines for regulation of chemicals are also within its scope.
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