Animal weights and skin fold thickness

All animals consumed the three diets and gained weight. Male mice were given diets to provide 5 g food/day while female were given 4 g/day, and mice were allowed to consume as much of this allotment as desired. Male mice were significantly heavier than female mice (P < 0.001). Within a sex, there were no significant differences in body weights of animals on the different diets, or with UV exposure. Others have also found that rodents who consume tomato diets are heavier than mice on purified AIN-93G diets [27] while some have found no difference [21]. Skin fold thickness 48 hours after the first exposure to UV radiation can be used as a rough measurement of the inflammation response. Male mice had significantly thicker skin than female mice (P < 0.001), with an average (± standard deviation) thickness of 1.23 ± 0.15 mm in males as compared to 1.02 ± 0.11 mm in females. This held true even in mice not exposed to UV denoting that there are fundamental differences in the physiology of skin between the sexes. There was no significant differences in skin fold thickness between the control and tomato diets for either sex after exposure to UV, consistent with previous reports from our group (data not shown)14.

Carotenoid profile in tomato diets

The control, tangerine and red tomato diets were extracted and analyzed for carotenoids (Table 1). The control diet did not contain tomato or any carotenoids. Phytoene and phytofluene were present in approximately twice the concentration in the tangerine tomato diet compared to the red tomato diet. Total lycopene was approximately three times higher in the red tomato diet compared to the tangerine diet. About 1% of the lycopene in the tangerine tomato diet was present as all-trans-lycopene while 82% of the lycopene in the red tomato diet was present as all-trans. Overall, the tangerine tomato diet had approximately a 20% higher level of total carotenoids compared to the red diet. ζ-carotene, neurosporene and tetra-cis-lycopene were absent in the red tomato diet, and β-carotene was absent in the tangerine tomato diet. Diets were formulated based on equal weight addition of tomato powder and are therefore not matched for any carotenoid levels.

Table 1 Carotenoid composition of tangerine and red tomato fortified dietsa. Full size table

Carotenoid levels in blood plasma and skin

Carotenoids exist in the plasma of mice fed both tomato diets in the mid-to-high nanomolar range, with lycopene being the most prevalent carotenoid present. Statistically significant differences in carotenoid levels between mice by UV status, sex and tomato diet are noted for plasma in Table 2 and for skin in Table 3. Phytoene and phytofluene were found to be significantly higher in the blood of mice fed tangerine tomatoes (P < 0.001 for both carotenoids), as well as higher in females as compared to males (P < 0.0001 and P = 0.0102, respectively). ζ-Carotene, neurosporene and tetra-cis-lycopene are absent in the animals fed red tomato diets (given they are absent in red tomatoes), and therefore significantly higher in animals fed tangerine tomato containing diets. Other-cis-lycopene is significantly higher in mice fed tangerine tomatoes (P < 0.001) while all-trans-lycopene (P < 0.0001) and 5-cis-lycopene (P < 0.0001) are higher in mice fed red tomato diets. However, there was no overall significant difference in total plasma lycopene concentrations between the two diets. Since there was approximately 3 times less total lycopene delivered in the tangerine tomato diet, which still resulted in similar plasma lycopene levels, lycopene from tangerine tomatoes appears to be considerably more bioavailable compared to red tomatoes. This is consistent with what our group has seen in post-prandial bioavailability studies in humans, with higher bioavailability of lycopene from tangerine tomatoes as compared to red15.

Table 2 Concentration of carotenoids in plasma of mice consuming tangerine and red tomato diets. Full size table

Table 3 Concentration of carotenoids in murine skin fed diets containing tangerine and red tomato powders. Full size table

Carotenoids were found in the skin of mice in mid-picomole to low nanomole per gram concentrations (Table 3). Phytoene, all-trans-lycopene plus other-cis-lycopene and total lycopene content in skin was affected by sex, with females accumulating higher levels than males. Phytofluene, ζ-carotene, tetra-cis-lycopene and total lycopene were significantly higher in mice fed tangerine tomato diets, while phytoene and all-trans plus other-cis-lycopene were not different between the two diets. Because of significant matrix suppression with quantiation by MS (signal too low for PDA) for many carotenoids using C30 stationary phases, we chose to move to a C18 stationary column, which chromatographed interfering skin lipids separately from carotenoids, allowing quantitation via MS without the ~10-fold matrix suppression observed using C30 columns. However, this move from C30 to C18 eliminated more detailed data about lycopene isomer profiles in the skin, as here we report tetra-cis-lycopene and a combination of all-trans-lycopene plus other non-tetra-cis, cis-lycopene isomers as compared to tetra-cis-lycopene, other-cis-lycopene, all-trans-lycopene and 5-cis-lycopene quantitated separately in the plasma.

Carotenoid levels in both plasma and skin were of similar magnitude as found by Kopec et al. where 10% tangerine tomato diets were fed for 10 weeks, mice were exposed to a single dose of UVB light (1 M.E.D.) and scarified 48 hours later14. Lycopene was the carotenoid present in highest concentration in both plasma and skin in both tomato diets, despite not being the most prevalent carotenoid in the tangerine tomato diet. Hata et al. found an average of 69 ng lycopene (129 pmol lycopene/g) and 65 ng phytoene (120 pmol phytoene/g) per gram skin, in the range of what we see in the SKH-1 mice. Ribaya-Mercado found approximately 1.5 nmol lycopene/g skin as determined using HPLC while Mayne et al. found from 100–800 ng carotenoids/g skin (approximately 186–1493 pmol carotenoid/g skin) as determined using Raman resonance spectroscopy16, 17. This data suggests that SKH-1 mice accumulate carotenoids in skin in reasonably similar concentrations as do humans, contrary to what is seen in blood plasma. Lycopene has been shown in cell culture to follow a U-shaped curve in terms of its benefit in fibroblasts exposed to UVB light. At optimal levels (0.05 nmol/mg protein), lycopene was shown to decrease UV-induced formation of thiobarbaturic acid-reactive substances (TBARS) to 40–50% the levels of controls, indicating protection against lipid peroxidation18. A study in hairless mice found that supplementation of diets with palm fruit carotenoids can decrease TBARS from controls19. There were no significant differences due to UV status, likely because carotenoids were measured at the end of study, 15 weeks after the last UV treatment. Others have found that UV radiation decreases carotenoids in skin (but not plasma) when skin is collected 48 hours after a one-time exposure20. A similar result has been observed in humans, where reductions in plasma carotenoids were noted after individuals were exposed to UVA and UVB light over a two week period21. The possibility exists that our skin samples were taken too long after the last UV exposure (15 weeks) and by this point, UV induced differences in carotenoid content in the skin or plasma are lost.

There was considerable inter-individual variability, consistent with the use of an outbred strain of mouse. Large heterogeneity is consistent with human populations but requires large numbers of animals for studies to be powered to see differences between treatments. Rodents tend to not be very good models for carotenoid absorption and distribution since they do not absorb carotenoid intact unless they are fed at supra-physiological doses22. In this study, the dose of tomato given to the mice was chosen to be sufficient to produce plasma lycopene levels that are consistent with humans consuming a diet rich in lycopene containing foods. Studies in free-living humans have shown a range of lycopene in plasma, from 200–1000 nmol/L23, 24. Here, continued consumption of diets containing 10% by weight tomato powder, mice reached 250–500 nmol/L lycopene. This again provides additional evidence that carotenoid bioavailability and metabolism differs between mice and humans.

Despite three times higher concentrations of phytoene in the tangerine tomato diet, lycopene was present at 10 fold higher levels in skin, and 2–4 fold higher concentrations in plasma. This provides rationale to feeding tangerine tomatoes when increasing tissue lycopene is desirable, and contrary to others who have found that phytoene is more bioavailable than lycopene25, 26.

Tumor incidence

To our knowledge, this is the first study investigating the effects of tomato consumption on keratinocyte carcinomas in vivo. Tumor number from 20 to 35 weeks can be seen in (Fig. 1). Numbers below are means ± SEM. Male mice on control diets developed significantly more tumors (4.04 ± 0.65 tumors) than male mice on red tomato diets (1.73 ± 0.50 tumors, P = 0.015). Male mice on control diets developed significantly more tumors than mice on tomato-containing diets (i.e. both red and tangerine tomato fed mice combined, 2.03 ± 0.45 tumors, P = 0.017). These data provide convincing evidence that tomato consumption, in a model of disease prevention, can modulate risk for cutaneous squamous cell carcinomas. Male mice on control diets were not significantly different from male mice on tangerine diets (2.36 ± 0.50 tumors, P = 0.2197). There were no significant differences in tumor number for any of the female mice. Previous studies have demonstrated that after exposure to UVB, male mice develop tumors earlier, and these tumors are more numerous, larger and more aggressive than in female SKH-1 mice27. These changes may also be attributable to lower antioxidant levels, more cutaneous oxidative DNA damage and increases in GR-1+CD11b+ myeloid cells in males27, 28. We hypothesize that because the overall number of tumors were small, differences were not noted between diet groups in female mice, though this would require additional experiments to demonstrate. No significant differences of tumor grade were found among any of the treatment groups. Feeding 10% tomato containing diets has been shown to decrease tumor development at other sites, including prostate29, 30. Previous work has demonstrated that feeding a 10% tangerine tomato containing diet can decrease UV-induced production of cyclobutane pyrimidine dimers, myeloperoxidase activity and p53 positive epidermal cells in male SKH-1 mice, suggesting that tomatoes may act via reducing inflammation and subsequent DNA damage in the skin14. It has also recently been shown that administration of a lycopene rich tomato nutrient complex could be protective against UVA1 (longwave UV radiation) and associated alterations in gene expression31. Other compounds (including melatonin and vitamin D metabolites) have also been demonstrated to be protective against UVB light32, 33.

Figure 1 Tumor number progression in male mice fed control diets (dotted line) vs. tangerine tomato diets (dashed line) vs. red tomato diet (solid line). Significant differences exist at end-of-study between animals on red vs. control diets, and on tomato (both diets pooled) vs. control diets. Full size image

Untargeted metabolomic analyses

Each carotenoid seems to have little relationship between concentration in the plasma and skin after linear regression (R2 < 0.1) suggesting, in this model, that skin carotenoid analysis would be necessary to understand concentrations in the skin itself. Additionally, when regressing tumor number with skin or plasma carotenoid concentrations, little relationship is apparent (i.e., mice with higher plasma or skin carotenoids do not seem to have fewer or smaller tumors). This lead us to conduct untargeted metabolomic analyses of murine skin, to better understand the global differences in small molecular weight metabolites present, without any preconceived bias.

Using untargeted metabolomics allows global profiling of differences between groups; in this case, the skin of animals fed control or tomato-containing diets. After removal of compounds present in processing blanks, full scan MS data collection on methanol extracts yielded over 8000 entities, with the stipulation that each entity must be present in every sample in at least one group. After conducting moderated t-tests (between animals on control vs. tomato diets) or ANOVA (comparing animals on control vs. tangerine tomato vs. red tomato) with Tukey’s post-hoc test and applying the Benjamini-Hochberg false discovery rate multiple testing correction, 17 entities achieved statistical significance. Of these 17 compounds, 10 can be definitively or tentatively identified as derived from tomato glycoalkaloids (Table 4).

Table 4 Metabolites in murine skin that differentiate animals on control vs. tomato diets linked to tomato glycoalkaloids. All compounds listed are absent in control animal skin and present in skin of animals on both tangerine and red tomato diets. Full size table

Tomatitidine, the aglycone of tomato glycoalkaloid α-tomatine, was confirmed to be present via authentic standard. Both tomatidine and α-tomatine have been shown to be anti-carcinogenic in vitro 34, 35 and have in vivo biological effects (including inhibiting skeletal muscle atrophy and weakness36, 37 and reducing plasma lipoprotein concentration38), lending plausibility to the idea that these compounds could be at least partially responsible for the decreases in tumor number observed here. Other identities can be tentatively assigned via accurate mass, relative retention time and MS/MS fragmentation patterns. This represents the first report of derivatives of tomato glycoalkaloids in vivo, as these compounds were previously thought to not be absorbed39, 40. The lack of absorption of tomato glycoalkaloids was the proposed reason for their lack of toxicity observed in populations consuming high glycoalkaloid tomatoes41.