Neurobiology Laboratory, Department of Zoology, School of Life Sciences, Ravenshaw University, Cuttack, 753003, India.
In Vitro Cell Dev Biol Anim. 2019 Sep;55(8):665-675. doi: 10.1007/s11626-019-00378-9. Epub 2019 Jul 10.
Benzo[a]pyrene (B[a]P) is an ubiquitous environmental pollutant that is generated during combustion of fossil fuels. We examine the effect of noradrenaline (NA) on B[a]P-induced neurotoxicity in brain tumor cell lines like neuroblastoma (Neuro2a) and glioma (C6). We pre-treated tumor cells with NA for 6 h, followed by addition of B[a]P for additional 24 h. Cell viability was measured using trypan blue dye-exclusion assay and comet assay was performed to measure DNA damage. Cell cycle status was analyzed using flow cytometry and oxidative DNA damage (8-oxodG) production was examined by immunostaining. The intracellular Ca concentration was analyzed using Fura-2AM. Our results showed viability of Neuro2a and C6 cells declined (24% and 20%) in B[a]P-treated groups. However, pre-treating with NA increased viability of cells by reducing percentage of cell death in both. Furthermore, B[a]P-induced deregulation of cell cycle (G2/M and S phase cell arrest) was significantly restored by pre-treatment with NA in Neuro2a cells as compared to C6 cells. We further observed increased 8-oxodG production in B[a]P-treated cells; however, NA pre-treatment significantly (p < 0.05) reduced the 8-oxodG production in Neuro2a, while C6 cells were less affected possibly due to better protective machinery. B[a]P-induced intracellular Ca influx was significantly reduced in both the cell lines due to co-treatment of NA possibly by reducing Ca influx. NA protects brain tumor cells against B[a]P-induced neurotoxicity may be by decreasing percentage of G2 cell arrest, oxidative DNA damage, and reducing intracellular Ca influx. These findings suggested that NA may be considered as a natural potential protective agent against B[a]P-induced neurotoxicity. Graphical abstract Graphical abstract showing differential protective mechanism of NA against B[a]P-induced toxicity through antioxidant mechanism maintaining homeostasis for oxidative stress in Neuro2a and C6 cell lines. The schematic graph showed the biological significance of the NA that regulates the induction of metabolic processes of cell cycle after exposure to the environmental pollutants. B[a]P increases the intracellular levels of Ca, but also induces damage to cellular molecules including DNA causing cell cycle arrest. The B[a]P-induced DNA damage due to base lesions generated in the genome, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is one of the most abundant because of guanine's lowest redox potential among DNA bases through intracellular calcium homoeostasis.
苯并[a]芘（B[a]P）是一种普遍存在的环境污染物，在化石燃料燃烧过程中产生。我们研究了去甲肾上腺素（NA）对神经母细胞瘤（Neuro2a）和神经胶质瘤（C6）等脑肿瘤细胞系中 B[a]P 诱导的神经毒性的影响。我们先用 NA 预处理肿瘤细胞 6 小时，然后再加入 B[a]P 24 小时。使用台盼蓝染料排除试验测量细胞活力，并用彗星试验测量 DNA 损伤。通过流式细胞术分析细胞周期状态，并用免疫染色法检测氧化 DNA 损伤（8-氧鸟嘌呤）的产生。使用 Fura-2AM 分析细胞内 Ca 浓度。我们的结果表明，B[a]P 处理组的 Neuro2a 和 C6 细胞活力下降（分别为 24%和 20%）。然而，NA 的预处理通过减少两种细胞的细胞死亡百分比，增加了细胞活力。此外，与 C6 细胞相比，NA 预处理显著恢复了 B[a]P 诱导的细胞周期失调（G2/M 和 S 期细胞阻滞）。我们进一步观察到 B[a]P 处理的细胞中 8-氧鸟嘌呤的产生增加；然而，NA 的预处理显著（p<0.05）降低了 Neuro2a 中的 8-氧鸟嘌呤的产生，而 C6 细胞受影响较小，可能是因为它们具有更好的保护机制。由于 NA 的共同处理，B[a]P 诱导的细胞内 Ca 内流在两种细胞系中均显著减少，可能是通过减少 Ca 内流。NA 可能通过降低 G2 细胞阻滞、氧化 DNA 损伤和减少细胞内 Ca 内流来保护脑肿瘤细胞免受 B[a]P 诱导的神经毒性。这些发现表明，NA 可能被认为是一种天然的潜在保护剂，可预防 B[a]P 诱导的神经毒性。
