Carotenoid/retinoid Modulation Of Cellular Redox Status

最近的预防和干预试验的结果表明，在疾病状态的早期食用富含抗氧化剂的食物具有保护优势，而后期通过饮食类胡萝卜素/类维生素a进行干预可能会加剧癌症。我们正在进行一系列基于机制的假设驱动研究，旨在解释在人类干预试验中观察到的矛盾结果。我们假设增加的抗氧化细胞环境（通过高水平的饮食补充化学抗氧化剂n -乙酰- l-半胱氨酸（NAC）或类胡萝卜素/类维生素a提供）将改变细胞氧化还原状态并促进致癌物引发小鼠的癌症。到目前为止，我们已经观察到膳食NAC增加了生存率，减少了皮肤恶性肿瘤的多样性，但增加了局部给药B诱导的恶性肿瘤的比例[a]P。大多数恶性肿瘤（角膜棘层瘤、鳞状癌和梭形细胞瘤）诱导FVB/N-p53缺陷Tg。AC小鼠表现出突变的转基因ras表达，p53核阳性（强度从0到+4区域不等），与治疗无关。无论是转基因表达阴性还是阳性的恶性肿瘤，内源性c-Ha-ras原癌基因的密码子12和密码子61突变均为阴性。NAC单独对脾细胞有丝分裂作用。目前，我们正在进行一项生命研究（γ射线照射FVB/ n杂合p53+/-小鼠，剂量分别为0、2、4或6 Gy），并在饮食中添加和不添加NAC。我们观察到，与4 Gy + NAC相比，对照组24周后（0/15 vs 10/15）胸腺淋巴瘤潜伏期降低，胸腺淋巴瘤发病率增加。体外研究表明，脂多糖（LPS，一种b淋巴细胞有丝分裂原）诱导的脾细胞增殖（T细胞与b细胞的比例为3:1）被NAC显著增加，但细胞凋亡被显著抑制，至少部分解释了这些结果。通过独立检测（TUNEL和Annexin V）证实了细胞凋亡的抑制作用。采用B淋巴细胞特异性单克隆抗体和t淋巴细胞特异性单克隆抗体，流式细胞术证实B淋巴细胞特异性。当目前的组织病理学研究完成后，我们将重点研究抗氧化加剧淋巴瘤形成的分子机制。这些研究将包括mRNA和蛋白水平的基因表达变化，以及增殖和凋亡关键信号通路的分子遗传变化。此外，体外研究将集中在1)使用基于微阵列的基因表达NAC相关的凋亡抑制机制，随后使用蛋白2D凝胶电泳和western分析进行确认（在需要确认失调通路的地方）。

Results from recent prevention and intervention trials imply that consumption of antioxidant-rich foods early in disease states provides a protective advantage, while late intervention with dietary carotenoids/retinoids may exacerbates cancer. We are conducting a series of mechanism-based hypothesis driven studies designed to explain the paradoxical results observed in human intervention trials. We hypothesize that increased antioxidant cellular environment (provided by high levels of dietary supplementation with the chemical antioxidant N-acetyl-L-cysteine (NAC), or carotenoids/retinoids, will alter the cellular redox state and promote cancer in carcinogen initiated mice. Thus far, we have observed that dietary NAC increases survival and reduces multiplicity of skin malignancies but increases the fraction of malignancies induced by topically administered B[a]P. The majority of the malignancies (keratoacanthomas, squamous carcinomas, and spindle cell tumors) induced in FVB/N-p53 deficient Tg.AC mice demonstrated mutant transgene ras expression and were nucleus positive for p53 (variable intensity from 0 to +4 areas) and treatment independent. Malignancies, both negative and positive for transgene expression were also negative for mutations in codon 12 and codon 61 of endogenous c-Ha-ras protooncogene. NAC alone is mitogenic to splenocytes. Currently, we are conducting an in life study (gamma irradiated FVB/N-heterozygous p53+/- mice at 0, 2, 4, or 6 Gy) with and without NAC supplementation in the diet. We have observed a decreased latency for thymic lymphoma and an increased incidence of thymic lymphoma after 24 wks (0/15 vs 10/15) in the controls versus 4 Gy plus NAC. These results may be explained, at least in part, by in vitro studies that show that lipopolysaccharide (LPS, a B-lymphocyte mitogen) induced splenic cell proliferation (3:1 T- to B-cell ratio) is significantly increased by NAC, but apoptosis was significantly suppressed. Suppression of apoptosis was confirmed by independent assays (TUNEL and Annexin V). B-lymphocyte specificity was confirmed by flow cytometry using B- and T-lymphocyte specific monoclonal antibodies. When current post- and in-life studies are completed for histopathology, we will focus on investigation of the molecular mechanisms involved in antioxidant exacerbation of lymphomagenesis. These studies will include changes in gene expression at the mRNA and protein level as well as molecular genetic changes in signaling pathways critical to proliferation and apoptosis. In addition, in vitro studies will be focused on 1) mechanism of NAC associated suppression of apoptosis using microarray based gene expression, followed by confirmation using protein 2D gel electrophoresis and western analysis (where required for confirming dysregulated pathways).