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Goodwin Ave. Dorner Dr. Progress in learning about underlying carotenoid bioactivity mechanisms has been limited due to lack of commercially available radiolabeled lycopene LYC , phytoene PE , and phytofluene PF. Tomato Solanum lycopersicum cv. To increase carotenoid production, two bleaching herbicides were administered during the culture incubation, 2- 4-chlorophenyl-thio triethylamine and norflurazon separately or in combination to produce varying ratios of PE, PF, and LYC.

Treatment with both herbicides resulted in optimal production of all three carotenoids. Adding [ 14 C] sucrose on day 1 of the d culture incubation cycle to norflurazon-treated cultures led to a small increase in labeling efficiency compared to adding it on day 7. Improved culture conditions efficiently provided sufficient 14 C-carotenoids for future cell culture and animal metabolic tracking studies.

A substantial body of epidemiological evidence suggests that tomato consumption is associated with a decreased risk of a variety of cancers including lung, stomach, and prostate with the strongest evidence for prostate cancer 1 , 2. Tomatoes provide many nutrients to the diet including carotenoids which are 40 carbon, pigmented compounds that range from colorless to red, yellow, and orange 3.

The most predominant carotenoid in red tomatoes is lycopene LYC , followed in concentration by phytoene PE , phytofluene PF , beta-carotene, alpha-carotene, and lutein and zeaxanthin 3. These prominent carotenoids are believed to be critical to the efficacy of tomatoes to prevent disease, however the majority of disease prevention research focuses on LYC alone.

To best understand the underlying mechanisms of prostate cancer risk reduction afforded by tomato consumption, a number of investigations into the bioavailability, biodistribution, and metabolism of tomato carotenoids have been undertaken 4 — 9. Plant cell culture is a useful method for radiolabeled bioactive compound production offering several advantages compared to whole plant biolabeling, such as the ability to target selected tissue sources based on desired secondary metabolite profiles, shortened culture periods, relative ease of extraction, and uniform exposure to isotopically labeled carbon sources in the media 10 — In order to enhance in vitro carotenogenesis, bleaching herbicides have been utilized 14 — Bleaching herbicides interrupt normal carotenogenesis either in vivo or in vitro , causing a fatal loss of photoprotection in the plant, leading to chlorophyll destruction upon light exposure VFNT cherry cells, and the tracers were used for in vitro investigation of their uptake into prostate cancer cells.

Further, NORF causes a concomitant increase in PDS expression as well as a modest increase in phytoene synthase PSY expression, likely due to increased photooxidative stress or a lack of end-product regulation of carotenogenesis Figure 1 The bleaching herbicide 2- 4-chlorophenyl-thio triethylamine CPTA has also been widely used in in vitro tomato research for the study of LYC accumulation and ripening mechanisms 16 , 17 , Another goal of this study was to enhance the radiolabeling efficiency of targeted carotenoids.

Plant secondary metabolites are produced during the growth plateau phase of cell culturing Therefore, examining different [ 14 C]-sucrose-dosing times during the growth cycle may lead to targeted enrichment of carotenoids. Suspension cultures were initiated and maintained according to a previously-described method using tomato cv.

VFNT cherry calyces for callus initiation Friable callus was transferred from agar-solidified media to solution media and subsequently transferred to fresh media every two weeks using published media formulations and culturing methods To alter the carotenoid biosynthetic pathway, thus optimizing specific carotenoid accumulation, cell cultures were treated with herbicides.

Based on previous work, herbicide treatments were as follows: cultures were either aseptically treated on day 1 with filter-sterilized aqueous CPTA To safely and efficiently 14 C label carotenoids from tomato cell suspension cultures, cells were subcultured into a reduced volume of media 72 mL , and 8 mL fresh media containing 0. Cultures were grown in a previously described enclosed Plexiglass radiolabeling chamber equipped with NaOH traps and an air-flushing system to prevent escape of 14 CO 2 from the growth chamber In CPTA-treated tomato cv.

VFNT cherry cells, the majority of LYC is produced after the end of growth, and continues to increase in concentration up to 32 days after inoculation Preliminary studies data not shown , indicated that when tomato cv. VFNT cherry cultures treated with CPTA were harvested at 14, 21, and 28 days, LYC concentration increased over time; however, harvest mass drastically decreased, resulting in overall lower LYC yield at 21 and 28 d compared to a d culture duration.

For this reason a d growth period was selected for further studies. It was hypothesized that adding [ 14 C]-sucrose on day 7 of the growth cycle rather than initially on day 1 would lead to increased 14 C-enrichment of PE and PF in NORF treated cultures. Three, d long [ 14 C]-radiolabeling runs were conducted. Filtration was ended and cultures were collected and weighed when no further liquid was expressed from the funnel for 30 s.

When cultures were collected from the radiolabeling chamber, samples were also taken from the NaOH traps and spent media for determination of cell labeling efficiency. Labeled and non-labeled carotenoids were extracted from frozen tomato cells as previously described 14 by using 0. The carotenoid-containing hexane phase was removed and the hexane extraction and centrifugation was repeated two more times. Roughly equivalent amounts of carotenoids generally between — ng were then repurified using the method described earlier using a C30 column and the HPLC-PDA system.

Carotenoid fractions were collected according to characteristic retention times, quantified based on authentic standards, and solvents were removed under argon. Tomato cells 0. Samples were held at room temperature for 45 min, scintillation cocktail was added, and then kept in darkness overnight; 14 C content was analyzed by scintillation counting the following day.

Carotenoid yields, however, were enhanced by herbicide treatments. Control cultures had substantially lower total carotenoid yield 0. Effects of herbicide treatments on production of three carotenoids from tomato cv.

VFNT cherry cell culture grown for d. See method section for details of treatments. Yield of minor carotenoids following herbicide treatment of tomato cv. VFNT cherry cell cultures after a d growth period. Data are averages of 2 trials, each analyzing 3 flasks, in duplicate. Eighteen percent of the initial 14 C dose accumulated in tomato cells and an estimated 0.

The specific activity of the carotenoids analyzed ranged from 0. Phytoene was the most abundant source of radiolabeled carotenoid 2.

To examine the effect of [ 14 C] sucrose dose administration timing on carotenoid enrichment, labeled sucrose was added to NORF-treated cell cultures on either day 1 or day 7 of the d growth cycle.

Dosing on day 1 vs. Labeled carbon distribution throughout experimental compartments was similar between the two radiolabeling runs Figure 5.

Carotenoid specific activity A and 14 C distribution in compartments of cultures treated with [ 14 C]-sucrose on day 1 full growth period B or day 7 of the growth period day 7—14 C. The use of successive CPTA day 1 and NORF day 7 treatments in a continuous growth cycle allows for the most robust production of a useful mixture of carotenoids for subsequent radiolabeling studies. In contrast, single herbicide treatments would be more effective in maximizing the yield of specific [ 14 C]-labeled carotenoids.

Bleaching herbicides increase total carotenoid content in tomato, pepper, and daffodil plants as well as tomato cell cultures 14 , 16 , 17 , 19 , 20 , 24 — The results from the current study confirmed either or both herbicides led to a greater total carotenoid yield than untreated cultures — fold. For NORF treatment, this has been attributed to either an upregulation of the metabolic pathway to compensate for a lack of photoprotective carotenoids or increased photostability of carotenoid precursors compared to their downstream products with longer polyene chains The findings from the current report support the first hypothesis, but not the second.

In the current study work, cell culture experiments were carried out in darkness where photostability would not be a contributing factor in carotenoid accumulation. With regards to increased carotenoid accumulation in tomato cultures with CPTA treatment, this may occur because of an upregulation of carotenogenesis in response to decreased BC or its derivatives, which may normally feed-back inhibit upstream carotenogenic enzymes The results did not support this hypothesis, showing instead that the combined and CPTA alone treatments resulted in similar total major carotenoid content.

Lastly, different herbicide treatments led to different mixtures of carotenoids. This novel method of producing carotenoids was thus chosen for subsequent radiolabeling studies. Tailored mixtures, predominant in one or another of the target carotenoids, could be encouraged by further alterations of the herbicide treatment. The current studies led to several improvements to the previously established carotenoid radiolabeling system Previously, harvest yield obtained from tomato cv.

This difference was likely due to adaptation of the cultures over time, in that the tomato suspension cultures had been continuously cultured over a period of 12—24 mo longer than the time of previous experimentation. Productivity differences between experiments could be responsible for the higher carotenoid yields and lower enrichments of the carotenoids, such that more 14 C was channeled into other biosynthetic pathways than carotenogenesis. Contrary to the hypothesis, adding [ 14 C]-sucrose later in the growth period did not significantly enhance the specific activity of PE and PF, but instead led to lower 14 C enrichment.

It is concluded that tomato cells should be grown with [ 14 C]-sucrose for the full growth period, and that the combined CPTA and NORF herbicide treatment induces the production of all three major tomato carotenoids. The methods described here for increasing total carotenoid yield, improving carotenoid profiles, and increasing carotenoid specific activity along with a recently improved tomato cell extraction protocol 28 , enhance the efficient production of radiolabeled carotenoids.

In addition, these results demonstrate that changing herbicide treatments can alter carotenoid profiles. One future direction of interest would be to use a high LYC producing tomato cell line to further increase overall carotenoid-producing potential. The methods described here could also be used for producing stable isotope labeled tracers for human metabolic research, using [ 13 C]-glucose as the primary carbon source for the cultures.

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