Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, US FDA, Jefferson, AR 72079, United States.
Office of Scientific Coordination, National Center for Toxicological Research, US FDA, Jefferson, AR 72079, United States.
Toxicol Sci. 2024 Sep 1;201(1):129-144. doi: 10.1093/toxsci/kfae070.
Lorcaserin is a 5-hydroxytryptamine 2C (serotonin) receptor agonist and a nongenotoxic rat carcinogen, which induced mammary tumors in male and female rats in a 2-yr bioassay. Female Sprague Dawley rats were treated by gavage daily with 0, 30, or 100 mg/kg lorcaserin, replicating bioassay dosing but for shorter duration, 12 or 24 wk. To characterize exposure and eliminate possible confounding by a potentially genotoxic degradation product, lorcaserin and N-nitroso-lorcaserin were quantified in dosing solutions, terminal plasma, mammary, and liver samples using ultra-high-performance liquid chromatography-electrospray tandem mass spectrometry. N-nitroso-lorcaserin was not detected, supporting lorcaserin classification as nongenotoxic carcinogen. Mammary DNA samples (n = 6/dose/timepoint) were used to synthesize PCR products from gene segments encompassing hotspot cancer driver mutations, namely regions of Apc, Braf, Egfr, Hras, Kras, Nfe2l2, Pik3ca, Setbp1, Stk11, and Tp53. Mutant fractions (MFs) in the amplicons were quantified by CarcSeq, an error-corrected next-generation sequencing approach. Considering all recovered mutants, no significant differences between lorcaserin dose groups were observed. However, significant dose-responsive increases in Pik3ca H1047R mutation were observed at both timepoints (ANOVA, P < 0.05), with greater numbers of mutants and mutants with greater MFs observed at 24 wk as compared with 12 wk. These observations suggest lorcaserin promotes outgrowth of spontaneously occurring Pik3ca H1047R mutant clones leading to mammary carcinogenesis. Importantly, this work reports approaches to analyze clonal expansion and demonstrates CarcSeq detection of the carcinogenic impact (selective Pik3ca H0147R mutant expansion) of a nongenotoxic carcinogen using a treatment duration as short as 3 months.
氯卡色林是一种 5-羟色胺 2C（血清素）受体激动剂和非遗传毒性大鼠致癌物质，在为期 2 年的生物测定中，它会导致雄性和雌性大鼠的乳腺肿瘤。雌性 Sprague Dawley 大鼠通过灌胃每天接受 0、30 或 100mg/kg 氯卡色林治疗，复制生物测定剂量，但持续时间更短，为 12 或 24 周。为了描述暴露情况并消除可能由潜在遗传毒性降解产物引起的混杂因素，使用超高效液相色谱-电喷雾串联质谱法在给药溶液、终末血浆、乳腺和肝脏样品中定量测定氯卡色林和 N-亚硝基氯卡色林。未检测到 N-亚硝基氯卡色林，支持氯卡色林被归类为非遗传毒性致癌物质。从包含热点癌症驱动突变的基因片段（即 Apc、Braf、Egfr、Hras、Kras、Nfe2l2、Pik3ca、Setbp1、Stk11 和 Tp53 基因）合成 PCR 产物，使用来自乳腺 DNA 样本（n=6/剂量/时间点）。使用 CarcSeq（一种经过错误纠正的下一代测序方法）对扩增子中的突变体分数（MF）进行定量。考虑到所有恢复的突变体，在氯卡色林剂量组之间未观察到显著差异。然而，在两个时间点都观察到 Pik3ca H1047R 突变的显著剂量依赖性增加（ANOVA，P<0.05），与 12 周相比，24 周时观察到更多的突变体和具有更大 MF 的突变体。这些观察结果表明，氯卡色林促进了自发发生的 Pik3ca H1047R 突变克隆的生长，从而导致乳腺肿瘤发生。重要的是，这项工作报告了分析克隆扩展的方法，并证明了 CarcSeq 检测非遗传毒性致癌物质（选择性 Pik3ca H1047R 突变体扩展）的致癌影响，使用的治疗持续时间短至 3 个月。
