2Colorado Biostatistics Consortium, University of Colorado Denver, Denver, CO, USA
Agricultural sciences rely almost entirely on chemical assays to screen the thousands of crop cultivars generated in a crop improvement program for potential human health benefits. This situation exists in part because most animal models are expensive to implement, utilize large amounts of plant material, and require specialized facilities and technical training. A cost-effective, high-throughput animal model to screen crop extracts for small molecules with biological activity related to human health benefits would provide a much-needed resource. Here we introduce a new, relatively high-throughput method incorporating the microscopic Caenorhabditis elegans nematode, which is suitable for screening chemical extracts for bioactivity without the need for robotics. This system was chosen because longevity extension in C. elegans has been previously associated with human health benefits. Through the use of cell culture inserts, C. elegans can be exposed to fresh crop extracts daily throughout their lifespan without mechanical manipulation of the worm, thus minimizing stress and creating an environment suitable for experiments measuring longevity. Additionally, the duration of longevity experiments can be reduced by using type II right censoring in experimental design and survival analyses.
Experimental manipulations that extend the longevity of an organism—whether it be yeast, fruit flies, the nematode Caenorhabditis elegans, various rodents, or nonhuman primates—also protect humans against a range of chronic diseases (1,2,3,4,5). Because of this, C. elegans has become widely used as a model for investigating longevity extension. This small nematode, approximately 1 mm in length, is one of the best-understood multi-celled organisms on the level of cellular development (6,7,8). Pharmaceutical companies currently utilize C. elegans to screen drugs for efficacy and toxicity (9,10,11,12,13,14), and environmental agencies are monitoring effects on gene expression when the nematode is exposed to xenobiotics, including fertilizers and pesticides found in field runoff (15).
In the agricultural sciences, chemical assays prevail for screening for potential human health benefits from the thousands of crop cultivars generated in crop improvement programs. This situation exists in part because most animal models are expensive to implement, utilize a large amount of plant material, and require specialized facilities and technical training. A cost-effective, high-throughput animal model to screen food crop extracts for small molecules with biological activity related to human health benefit would provide a much-needed resource. The approach evaluated in this study was to monitor the effects of food crop extracts on C. elegans longevity, given that longevity extension has been associated with human health benefits. It has recently been published that biological pathways that dictate aging and lifespan in yeast, nematodes, and humans are highly evolutionarily conserved (16), and that these processes are also influential in the progression of cancer and other age-related diseases (17,18,19,20,21). Moreover, pathways known to control longevity in C. elegans are also known to regulate critical processes of age-related diseases in humans (22,23,24).
The advantages of utilizing this nematode include its small size, the small amount of crop extract required for assays, the short duration of experimental protocols, and the minimal requirement for laboratory facilities. Here we describe the development of a C. elegans longevity assay to screen for biological activity of whole food phytochemical extracts utilizing the wild-type N2 Bristol strain. The assay utilizes cell culture inserts and liquid culture as a means to expose C. elegans to fresh food crop extracts daily, which eliminates the required transfer step of current methods (a process that can itself affect longevity due to mechanical manipulation of the nematode). We also evaluate the use of right censoring as a means to shorten study duration.Materials and methods Study design
The experiment was designed to compare the effects on longevity of 2 dry bean market classes plus control. In addition to the standard agar-based C. elegans longevity extension assay, a novel liquid culture feeding system was developed and tested with the same dry bean powders. Within the liquid culture feeding system, 3 different pH levels of hydrophilic and 1 hydrophobic fraction for each dry bean market class were tested, resulting in 8 distinct phytochemical delivery solutions, plus control. Extraction conditions can be found in the Supplementary Materials. Integrity of the extracts when stored at room temperature was monitored using LC-UV. Instrument setup and separation conditions are also located in the Supplementary Materials.C. elegans strain, maintenance and longevity assays
The wild-type N2 Bristol strain of C. elegans (Caenorhabditis Genetics Center, University of Minnesota, Minneapolis, MN, USA) was used. Worm stocks were maintained on nematode growth medium (NGM) plates seeded with 100 µL OP50 Escherichia coli (Caenorhabditis Genetics Center) as a food source at 20°C, as described (25).
In agar-based studies, deionized water bean extracts were added to a culture of OP50 E. coli at a concentration of 100 µg/mL and incubated 16 h at 37°C with shaking. E. coli with extract was added to 35 mm NGM-agar treatment plates containing cholesterol (10 µg/mL) and 25 µM 5-Fluoro-2′-deoxyuridine (FUDR; Cat. no. F0503; Sigma-Aldrich, Saint Louis, MO, USA) to suppress reproduction. Treatment plates were grown overnight at room temperature (22 ± 2°C), and synchronized young adult worms were added to a density of 20 ± 4 worms per plate (36–44 worms per treatment) (26). Worms were monitored 5 times per week for mortality and scored as dead if they failed to respond when gently prodded with a platinum pick.