Fujiishi Yohei, Ohyama Wakako, Okada Emiko
Yakult Central Institute, Yakult Honsha Co., Ltd., 5-11 Izumi, Kunitachi-Shi, Tokyo, 186-8650, Japan.
Genes Environ. 2024 Dec 18;46(1):26. doi: 10.1186/s41021-024-00320-w.
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.
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.
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.
在使用体外微核试验（MNT）评估含有益生菌候选菌株（如产乳酸细菌）的物质的遗传毒性时，测试培养基中的细菌生长可能会降低培养基的pH值。已知低培养基pH值会诱导细胞毒性和假阳性结果。在TK6细胞系统中，处理后通过洗涤很难完全从培养基中去除细菌，导致在短期处理的恢复期细菌生长。在本研究中，使用添加了乙酸、乳酸或甲酸（这些都是非遗传毒性细菌代谢产物）的培养基，研究了在TK6细胞MNT中产生假阳性的低pH范围。此外，为了使用抗生素（即青霉素/链霉素（P/S）、硫酸庆大霉素（GM）和两性霉素B（AP））抑制恢复期的细菌生长，确定了它们不影响TK6细胞生长或微核诱导的最大适用浓度。然后，我们使用一种含有活产乳酸细菌的物质进行了MNT，以验证抗生素的有效性。
在有和没有S9混合物的3小时处理中，乙酸分别在初始pH值≤6.2时、乳酸在初始pH值≤6.0时以及甲酸在初始pH值≤6.7时在连续处理中诱导TK6细胞产生微核（假阳性）。添加P/S、GM和AP的培养基在阴性和阳性对照中分别在≤400单位/毫升 - 400微克/毫升、≤250和≤20微克/毫升时不影响TK6细胞生长或微核化细胞频率。在含有活产乳酸细菌的发酵乳的MNT中，向恢复培养的培养基中添加P/S或GM可抑制细菌生长、降低pH值和细胞毒性。
本研究揭示了在短期和连续处理条件下TK6细胞MNT中产生假阳性的低pH范围。这些值将作为解释结果生物学相关性的参考。在短期处理中，恢复培养中最佳的抗生素添加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菌株的测试物质的遗传毒性。
