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Department of Dermatology, The George Washington University Medical Center, Washington, DC 20037; * Research Diets, New Brunswick, NJ 08901; and Biostatistics Center Medical Center Unit, The George Washington University Medical Center, Washington, DC 20037
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
FOOTNOTES
LITERATURE CITED
ABSTRACT 
Solar ultraviolet-B radiation (UVB; 290-320 nm) causes skin cancer and suppresses cell-mediated immunity, preventing the rejection of UV-induced tumors. One mechanism initiating UV suppression involves the trans to cis photoisomerization of urocanic acid (UCA), a histidine derivative found in the stratum corneum. The addition of L-histidine to nonpurified mouse diet has been shown to increase skin trans-UCA levels and sensitivity to UVB immune suppression. Specially formulated L-histidine diets (0.40-64 g/kg) fed to BALB/c mice that were monitored over a 19-wk period resulted in an unexpected modulation of skin trans-UCA. ANOVA revealed a group-time interaction, providing initial evidence that the skin levels of trans-UCA were modulating up and down in all groups except the control group (6.4 g/kg diet). We observed that both high (64 g/kg diet) and low (0.4 g/kg diet) levels of dietary L-histidine resulted in the increase of skin trans-UCA to levels significantly higher than those recorded in the control group. In mice fed these histidine levels, skin trans-UCA increased to between 2.9 and 3.6 nmol/mg skin (64 g/kg diet, over 5 wk; 0.4 g/kg diet, over 8 wk) and then decreased to ~1.69 nmol/mg skin, the base-line level (64 g/kg diet, over 11 wk; 0.4 g/kg diet, over 17 wk). The increase in trans-UCA levels in mice with low L-histidine intake may be the result of protein malnutrition, consistent with weight loss observed in those mice. The modulation of trans-UCA levels in skin by dietary L-histidine has not been previously described; its role in skin cancer development is under investigation.
KEY WORDS: ultraviolet-B radiation · urocanic acid · L-histidine · BALB/c mice · immunosuppressionINTRODUCTION
Solar ultraviolet-B (UVB4; 290-320 nm), the shortest rays of nonionizing sunlight able to penetrate to the earth's surface, cause malignant transformation of cells (Black and Chan 1977
, Forbes et al. 1978), and a selective systemic suppression of cell-mediated immunity (De Fabo and Noonan 1983, Noonan et al. 1981). UVB-induced immune suppression is a critical step in UV-carcinogenesis that prevents the immunologic rejection of highly antigenic UV-induced tumors (De Fabo and Kripke 1979, Fisher and Kripke 1982, Noonan and De Fabo 1992).
Our studies of the systemic suppression of contact hypersensitivity by prior UV irradiation are focused on understanding how UV radiation induces immune suppression. A previous experiment revealed a unique photoreceptor in skin that appears to control the initiation of the immune suppressive action of solar UVB (De Fabo and Noonan 1983b). The photoreceptor was identified as urocanic acid (UCA), which is formed on deamination of the essential amino acid L-histidine, via the catalytic action of the enzyme histidine-ammonia lyase (histidase, EC 4.3.1.3). Our working hypothesis for UVB-induced immune suppression proposes that absorption of UVB by trans-UCA leads to photoconversion to the cis isomer. cis-UCA initiates an "alteration" in antigen-presenting cells (APC, e.g., skin Langerhans cells, macrophages, lymph or spleen dendritic cells) such that antigen-specific down-regulatory T cells, rather than effector T cells, are formed as antigen is processed by the altered APC (De Fabo and Noonan 1983b, Greene et al. 1979). Evidence supporting this model continues to accumulate (see reviews in Noonan and De Fabo 1992, Norval 1996). We propose that, as the suppressor to effector T-cell ratio increases, down-modulation of an immune response against the sensitizing antigen occurs. If UV-induced skin tumor antigens are involved in skin malignancies (Hostetler et al. 1995), suppression of immune attack by cis-UCA against skin cells carrying these antigens is predicted to occur, allowing tumor outgrowth. It is this alteration of antigen-presenting cells and antigen processing by cis-UCA that we propose links UVB and UCA isomerization to systemic immune suppression and skin cancer. If this scenario is correct, the following question arises: can modified trans-UCA levels in the skin, formed in mice fed different dietary L-histidine, affect skin tumor development?
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Table 1. The L-amino acid-based diets used in this study1 |
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Table 2. One-way ANOVA to compare skin trans-UCA levels among all dietary groups at each time point1 |
One of our earlier studies showed that increased dietary L-histidine (100 g/kg nonpurified diet) leads to increased levels of mouse skin trans-UCA and increased sensitivity to UVB-induced immune suppression (Reilly and De Fabo 1991). This suggests that increased UVB immune suppression by L-histidine may play a role in skin cancer development.
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Table 3. Results from a first-order linear regression fitted to the mouse body weight data for each dietary group as shown in Figure 3 |
MATERIALS AND METHODS
(see Table 1). The specified amounts of L-histidine were added at the expense of cornstarch. Thus, the diets were not isonitrogenous, but all other nutrients were equal. To make the diets isonitrogenous, we would have had to add to our diet either a single nonessential amino acid or mix of such, which in either case would result in adding compounds with unknown consequences for UCA formation in the skin.
Feeding groups.
For 19 wk, five groups of 60 mice were fed the pelleted food (Table 1) containing the following levels of L-histidine: Group A, 0.4 g/kg diet; Group B, 1.6 g/kg diet; Group C (control group), 6.4 g/kg diet; Group D, 25.6 g/kg diet; and Group E, 102.4 g/kg diet. Five weeks into the experiment, it was noticed that the highest dose of L-histidine (Group E) was not well tolerated; at wk 10, this group was terminated because the mice continued to show severe signs of debilitation. Group E was replaced with a new group of mice fed a 64 g L-histidine/kg diet (Group F). This new level was well tolerated and was continued for a total feeding period of 19 wk.
UCA extraction.
Three mice were removed weekly from each feeding group for skin UCA level determination. The level of trans-UCA for all groups was measured at wk 0, before the customized diets were introduced. The mice were killed with halothane vapor and, under a yellow safe light (nonphotoisomerizing to UCA) that blocked UVA and UVB radiation to a level of less than 10
6 W/cm2, the dorsal surface of each mouse was shaved using electric clippers, exposing ~10 cm2 of skin. The shaved dorsal skin was dissected off, the subcutaneous fat removed by scraping with a scalpel blade and three punch biopsies (each 6 mm in diameter) were taken from each skin sample. The biopsy pieces from each mouse were wrapped together in preweighed aluminum foil to exclude any light, placed in a plastic bag on ice and transported to an analytical balance for weighing. The samples were then placed back under the yellow safe light, removed from the aluminum foil and the extraction protocol followed (see below).
Extraction protocol.
A modification of an extraction for UCA in human skin was used, as previously described (Jansen et al. 1991, Reilly and De Fabo 1991). In brief, each sample was diced finely with scissors into a 1.5-mL microcentrifuge tube (Fisher Scientific, Pittsburgh, PA) containing 100 µL of 1 mol/L KOH. Each sample tube was mixed with a vortex mixer and then placed on ice for a 10-min extraction period. The remainder of the extraction protocol was conducted under regular laboratory lighting (OCTRON 4100K 32W; Sylvania, St. Mary's, PA), and all samples were kept in the dark by covering with aluminum foil while on the bench top. Each sample tube was centrifuged for 5 min (16,300 × g) after which the supernatant was transferred to a clean tube, referred to as the extract tube. A second 100 µL of 1 mol/L KOH was added to the skin pellet and the procedure repeated. The skin pellet remaining in the sample tube was then washed with 200 µL of HPLC-grade distilled water (Fisher Scientific), the washings added to the extract tube and the extract neutralized with 200 µL 0.67 mol/L H3PO4. At this point, the covered extract tubes were placed at 
70°C. When all of the samples had been extracted for UCA and stored at 70°C, HPLC was carried out. Random sampling of the pellets remaining after extraction showed that no detectable levels of UCA could be found.
High performance liquid chromatography.
On removal from the 70°C freezer, the covered samples were thawed slowly at room temperature. The extract tubes were then centrifuged for 5 min (16,300 × g ) and 100 µL removed for analysis. Each 100-µL sample was diluted with 400 µL of acetonitrile and phosphate buffer (ACNP) [1 L ACNP = 800 mL HPLC-grade acetonitrile (Fisher Scientific) + 198 mL HPLC-grade distilled water + 2 mL phosphate buffer (49.0 mL 1 mol/L K2HPO4 + 51.0 mL 1 mol/L KH2PO4; pH = 6.8)]. Analysis was conducted using HPLC (Beckman System Gold Model 126, Beckman Bioanalytical Systems Group, Columbia, MD) with an Ultrasil-NH2 column (#235345). ACNP buffer was used as the eluant in isocratic phase. Purified cis-UCA (provided by Irma Santucci and John Finlay-Jones, Flinders University, Australia) and trans-UCA (Fisher Scientific), dissolved in ACNP (1.0 ng/µL), were used as standards. Throughout the procedure, great care was taken to minimize any exposure of the extracted tissue or extraction solution to room or window light to prevent isomerization of UCA (Hug and Hunter 1994) by keeping the tubes covered whenever possible.
Curve fitting.
The mean of trans-UCA values, at wk 0, is expressed in Figure 1 as a base line for comparative purposes. The lines plotted through the data points were calculated using a curve-fitting program (TableCurve, 2D v 3, Jandel, San Rafael, CA).
Body weights.
Each week, the mice remaining in the five feeding groups were weighed. The mice were placed in a preweighed container, in groups of up to 5, and weighed using an Ohaus triple beam balance (Florham Park, NJ). The average weight of the mice in each feeding group was calculated and used to monitor weekly changes in body mass. To assess the overall change in weight for Groups A-F throughout the experiment (19 wk), a first-order linear regression was fitted to the data and the F statistic used to indicate the significance of the slopes from zero at the level of P < 0.05.
Statistical analysis.
This study was designed as a two-factor factorial experiment, with time and dietary group as the two factors, in which three mice were used in each group for each week. Preliminary statistical analysis consisted of plotting the median trans-UCA level separately for each group (A-F) at each week over the 19-wk period. A LOWESS curve (Cleveland 1979), a locally weighted scatter plot smoother, was used to plot the trans-UCA level for three mice for each group at each week over the 19-wk period. A two-way ANOVA was used to analyze the data, and a significant group by time interaction was found. The five groups were compared at each week with a one-way ANOVA followed by the Scheffé multiple comparison procedure (Woolson 1987). The probability level at which differences were considered significant was P < 0.05.
RESULTS
DISCUSSION
found two components of histidine-ammonia lyase in partially purified liver extracts from normal mice. One showed 50% lower enzyme activity at high L-histidine concentrations. At low L-histidine concentrations, sigmoidal kinetics were observed. This in vitro study, although not identical to our in vivo results, indicates a complex interaction between histidase and its substrate, histidine, suggesting a concentration-dependent interaction that would be consistent with our findings.
). Because skin keratinization occurs in mouse skin, mechanisms controlling this reaction may be influenced by different concentrations of dietary L-histidine. Because L-histidine from filaggrin may be at least one of the precursors of trans-UCA in skin during keratinization (Scott et al. 1982), changes in dietary L-histidine may influence the concentration of filaggrin formed. This does not explain, however, the modulation of skin trans-UCA observed in our study. It does, nonetheless, leave open the possibility that keratinization may be influenced by changes in histidine intake.
, Whitehead 1964, Whitehead and Arnstein 1961).
, Forbes et al. 1978) and initiation of immune suppression (De Fabo and Noonan 1983a and 1983b, Noonan et al. 1981) play critical roles in photocarcinogenesis (De Fabo and Kripke 1979, Fisher and Kripke 1982, Noonan and De Fabo 1992). Dietary factors are also important in skin cancer formation (Black et al. 1992, Orengo et al. 1989, Reeve et al. 1988, Scotto and Fears 1987). The complete dietary ingredients used in this study are listed in Table 1. Although we are unaware of studies concerning effects of specific amino acids on skin cancer, certain unsaturated fatty acids, e.g., corn oil [(n-6) fatty acid source], have been shown to promote UV carcinogenesis in mice. Others, however, such as menhaden oil [(n-3) fatty acid source] protect against UV carcinogenesis (Black et al. 1985, Orengo et al. 1989). The mechanism involved appears to be related to the induction of prostaglandin E2, an immunosuppressive agent, by corn oil but not by menhaden oil. Taking this into consideration, the concentration of corn oil was constant in all diets used throughout these experiments (10 g/kg) (Fisher and Black 1991).
and 1983b, Noonan and De Fabo 1992). Therefore the question becomes, does modulation of trans-UCA by dietary L-histidine play a role in UV-induced skin cancer? We previously showed that upon exposure to UV radiation, trans-UCA in the skin photoisomerizes to cis-UCA, which initiates an antigen-presenting cell defect resulting in immunosuppression (Noonan et al. 1988). This form of immunosuppression appears to play a major role in subcarcinogenic doses of UV radiation preventing immunological rejection of UV-induced skin cancers (De Fabo and Kripke 1979).
) would be expected to increase. This, in turn, would lead to an increase in the formation of antigen-specific T-suppressor cells against the tumor antigen, via cis-UCA-induced alterations to antigen-presenting cell function as described previously (see Noonan and De Fabo 1992). Such interaction would allow the outgrowth of the malignantly transformed skin cells by preventing their immunologic rejection. Thus, continued exposure to UVB radiation, along with changing skin UCA levels caused by either high or low dietary L-histidine, may result in enhanced levels of tumor-specific suppressor T-cells and hence accelerated tumor outgrowth. We reported earlier that increased dietary L-histidine can lead to greater immune suppression for a given amount of UVB radiation (Reilly and De Fabo 1991). Others have reported direct association between the amount of exogenously applied trans-UCA and increased UV-induced skin cancer (Reeve et al. 1989). Whether changes in dietary L-histidine such as in this study will affect UV-induced skin cancer induction is currently under investigation.
ACKNOWLEDGMENTS
FOOTNOTES
Manuscript received 13 January 1997. Initial reviews completed 20 March 1997. Revision accepted 4 August 1997.
LITERATURE CITED
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