Shah Ume-Kulsoom, Seager Anna L, Fowler Paul, Doak Shareen H, Johnson George E, Scott Sharon J, Scott Andrew D, Jenkins Gareth J S
Institute of Life Sciences, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP, UK.
Safety & Environmental Assurance Centre (SEAC), Unilever, Bedford, MK44 1LQ, UK.
Mutat Res Genet Toxicol Environ Mutagen. 2016 Sep 15;808:8-19. doi: 10.1016/j.mrgentox.2016.06.009. Epub 2016 Jul 26.
Benzo[a]pyrene (B[a]P) is a known genotoxin and carcinogen, yet its genotoxic response at low level exposure has not been determined. This study was conducted to examine the interplay of dose and metabolic capacity on genotoxicity of B[a]P. Investigating and better understanding the biological significance of low level chemical exposures will help improve human health risk assessments. The genotoxic and mutagenic effects of B[a]P were investigated using human cell lines (AHH-1, MCL-5, TK6 and HepG2) with differential expression of the CYP450 enzymes CYP1A1, 1B1 and1A2 involved in B[a]P metabolism. MCL-5 and HepG2 cells showed detectable basal expression and activity of CYP1A1, 1B1 and 1A2 than AHH-1 which only show CYP1A1 basal expression and activity. TK6 cells showed negligible expression levels of all three CYP450 enzymes. In vitro micronucleus and HPRT assays were conducted to determine the effect of B[a]P on chromosome damage and point mutation induction. After 24h exposure, linear increases in micronucleus (MN) frequency were observed in all cell lines except TK6. After 4h exposure, only the metabolically competent cell lines MCL-5 and HepG2 showed MN induction (with a threshold concentration at 25.5μM from MCL-5 cells) indicating the importance of exposure time for genotoxicity. The HPRT assay also displayed linear increases in mutant frequency in MCL-5 cells, after 4h and 24h treatments. Mutation spectra analysis of MCL-5 and AHH-1 HPRT mutants revealed frequent B[a]P induced G to T transversion mutations (72% and 44% of induced mutations in MCL-5 and AHH-1 respectively). This study therefore demonstrates a key link between metabolic capability, B[a]P exposure time and genotoxicity.
苯并[a]芘（B[a]P）是一种已知的基因毒素和致癌物，但其在低水平暴露下的基因毒性反应尚未确定。本研究旨在探讨剂量与代谢能力对B[a]P基因毒性的相互作用。研究并更好地理解低水平化学物质暴露的生物学意义将有助于改进人类健康风险评估。使用参与B[a]P代谢的CYP450酶CYP1A1、1B1和1A2表达存在差异的人类细胞系（AHH-1、MCL-5、TK6和HepG2），研究了B[a]P的基因毒性和诱变作用。与仅显示CYP1A1基础表达和活性的AHH-1相比，MCL-5和HepG2细胞显示出可检测到的CYP1A1、1B1和1A2基础表达和活性。TK6细胞显示所有三种CYP450酶的表达水平可忽略不计。进行了体外微核试验和次黄嘌呤-鸟嘌呤磷酸核糖转移酶（HPRT）试验，以确定B[a]P对染色体损伤和点突变诱导的影响。暴露24小时后，除TK6外的所有细胞系中微核（MN）频率均呈线性增加。暴露4小时后，只有代谢能力较强的细胞系MCL-5和HepG2显示出MN诱导（MCL-5细胞的阈值浓度为25.5μM），表明暴露时间对基因毒性的重要性。HPRT试验在4小时和24小时处理后，MCL-5细胞中的突变频率也呈线性增加。对MCL-5和AHH-1 HPRT突变体的突变谱分析显示，B[a]P诱导的G到T颠换突变频繁（分别占MCL-5和AHH-1诱导突变的72%和44%）。因此，本研究证明了代谢能力、B[a]P暴露时间与基因毒性之间的关键联系。
