vervolg furocoumarine 4

Potapenko AIa, Malakhov M.V & Kiagova A.A.. (2004) "[Photobiophysics of furanocoumarins]"
Biofizika 49(2), 322-38. Abstract. Furocoumarins (psoralens) are photosensitizers of plant origin, which increase the sensitivity of biological objects to near ultraviolet (UV-A, 320-400 nm). In combination with UV-A, they are successfully used for treating many dermal and autoimmune
diseases (PUVA therapy and photophoresis). Along with therapeutic effects, the furocoumarin
photochemotherapy induces a number of side-effects (erythema, edema, hyperpigmentation, and premature aging of skin). All photobiological effects of furocoumarins result from their
photochemical reactions. Therefore, in order to advance the therapy, it is necessary to know the
photochemical mechanisms of induction of both side- and therapeutic effects. The types of
photoreactions of furocoumarins classified with respect to reactive photoproducts interacting with substrate were considered. Primary emphasis was placed on reactions proceeding with the
participation of photooxidation products of furocoumarins. Among these photoproducts, at least
two types can be distinguished. Some of them possess membranotoxic properties, others
produce the immunosuppressory action in vivo. The photochemical mechanisms of the formation
of the photoproducts of furocoumarins are different. It was found that, by varying the illumination conditions (intensity of UV-A radiation or the concentration of the photosensitizer), it is possible to obtain the photoproducts of furocoumarins that have either membranotoxic or
immunosuppressory properties. It was found that the mechanisms of the immunosuppressive
action of the photooxidation products of furocoumarins have some features in common with those underlying the PUVA therapy and photophoresis. It is assumed that the photochemical basis of the therapeutic action of furocoumarins is the reactions with the involvement of the products of their photooxidation.

Speight E.L. & Farr P.M. (1994) "Erythemal and therapeutic response of psoriasis to PUVA using
high-dose UVA" British Journal of Dermatology 131(5), 667. Abstract. In PUVA treatment of
psoriasis, clinical observation suggests that uninvolved skin is more susceptible to PUVA
erythema than lesions of psoriasis. If this is the case, then the efficacy of PUVA treatment might
be increased by using localized high-dose UVA restricted to lesional skin. We have therefore
studied the erythemal and therapeutic response of psoriasis to PUVA using high-dose UVA and,
for comparison, the erythemal response to UVB. In 14 patients, an area of psoriasis and adjacent oninvolved skin were exposed to a series of UVA doses (350 ± 30 nm, 1–16 J/cm2), using an irradiation monochromator. Six other patients were similarly phototested with a series of UVB doses (300 ± 5 nm, 20–112 mJ/cm2) to both uninvolved and lesional skin. Erythema was judged visually at 72 h for psoralen–UVA, and at 24 h for UVB, and measured using a scanning laser– Doppler velocimeter. In 10 patients, PUVA therapy using high-dose UVA was subsequently given to lesional skin (8–16 J/cm2 twice weekly) in addition to conventional whole-body PUVA. For psoralen–UVA, the minimal phototoxic dose within psoriasis was increased by a factor of 4 compared with non-lesional skin (P < 0.01, Wilcoxon signed-rank test). For UVB, the minimal erythema dose within psoriasis was higher than that for non-lesional skin (medians > 112 and 28 respectively, P < 0.05). Laser–Doppler measurements confirmed that the reduced erythemal sensitivity was not due to masking of response by pre-existing increased blood flux within psoriasis. In six patients, the sites subsequently treated twice weekly with PUVA, using high-dose UVA, cleared faster (median number of treatments 3), but with a similar cumulative UVA dose, compared with adjacent lesional skin treated with conventional PUVA (median number of treatments 12). This study demonstrates that psoriasis may clear rapidly, without burning, using high-dose UVA. Availability of a suitable irradiation apparatus would allow rapid and effective PUVA treatment to be used for localized, resistant disease.

Stern R.S., Laird N., Melski J., Parrish J.A., Fitzpatrick T.B. & Bleich H.L. (1984) "Cutaneous
squamous-cell carcinoma in patients treated with PUVA." N Engl J Med 310(18), 1156-61
Abstract. A 5.7-year prospective study of 1380 patients treated for psoriasis with oral
methoxsalen (8-methoxypsoralen) and ultraviolet A photochemotherapy (PUVA) revealed that
after adjustment for exposures to ionizing radiation and topical tar preparations, the risk that
cutaneous squamous-cell carcinoma would develop at least 22 months after the first exposure to PUVA was 12.8 times higher in patients exposed to a high dose than in those exposed to a low dose (95 per cent confidence interval, 5.8 to 28.5). No substantial dose-related increase was noted for basal-cell carcinoma. The dose-dependent risk of cutaneous squamous-cell carcinoma suggests that PUVA can act as an independent carcinogen. In our study, morbidity associated with these tumors has been limited, but further follow-up is needed. Meanwhile, patients treated with PUVA should be followed closely for the possible development of cutaneous squamous-cell carcinoma.

Stern R.S. & Lange R. (1988) "Non-Melanoma Skin Cancer Occurring in Patients Treated With
PUVA Five to Ten Years After First Treatment."Journal of Investigative Dermatology 91, 120–
124. Abstract. Continued prospective study of the 1,380 patients enrolled in the PUVA study for
10 years after first exposure to PUVA demonstrates a strong association between cumulative
exposure to PUVA and an increased risk of squamous cell carcinoma of the skin. For tumors
occurring at least 58 months after first treatment, after adjustment for age, sex, and area of
residence, we observed that patients with more than 260 treatments had an 11-fold increase in
risk compared to patients who had received 160 or fewer treatments during the same interval (P <0.01). Comparable increases in relative risk were noted in patients of all skin types, irrespective of prior ionizing radiation exposure. We also noted a modest dose-dependent increase in the risk for the development of basal cell carcinoma for patients who received an excess of 200 treatments compared to those who had received fewer than 160 treatments within the same time period (P <0.05). Tumors detected in our cohort exhibit biologic behavior similar to nonmelanoma skin cancers associated with sun exposure. Careful monitoring and early detection should limit the morbidity associated with these tumors.

Stern, R. S. (1994) “The carcinogenic risk of treatments for severe psoriasis.” Cancer 73, 2759-
2764. Abstract. BACKGROUND. Common treatments used for severe psoriasis include psoralen
and ultraviolet A radiation (PUVA), methotrexate, ultraviolet B (UVB), and tar. These therapies
are often used for prolonged periods and may be carcinogenic. METHODS. For more than 13
years, the authors have prospectively determined the incidence of skin cancer and use of
treatments for psoriasis in a 1380 patient cohort originally enrolled in a therapeutic trial of PUVA
at 16 university centers. RESULTS. Squamous cell carcinoma (SCC) developed in more than one
fourth of patients exposed to high doses of PUVA. In this group, the standard morbidity ratio for
these tumors was 83 (95% confidence interval [CI], 72-96) compared with the expected number
of these tumors in the general population. High-level exposure to methotrexate is a significant
independent risk factor for developing SCC (relative risk, 2.1 for high versus low or no exposure; 95% CI, 1.4-2.8). Metastatic disease developed in seven patients with SCC. No significant increase in the risk of SCC was associated with long term exposure to UVB or topical tar, and no substantial increase in the risk of basal cell carcinoma was noted in association with prolonged use of any of these treatments. CONCLUSIONS. Long term exposure to PUVA and methotrexate significantly increases the risk of SCC in patients with psoriasis. This risk should be considered in selection of treatment. The ultimate morbidity of these tumors is undetermined.

Stern R.S., Nichols K.T., Väkevä L.H. for The PUVA Follow-up Study (1997) "Malignant
Melanoma in Patients Treated for Psoriasis with Methoxsalen (Psoralen) and Ultraviolet A
Radiation (PUVA)" New England J of Medicine 336, 1041-1045. Abstract. Background
Photochemotherapy with oral methoxsalen (psoralen) and ultraviolet A radiation (PUVA) is an
effective treatment for psoriasis. However, PUVA is mutagenic, increases the risk of squamouscell skin cancer, and can cause irregular, pigmented skin lesions. We studied the occurrence of melanoma among patients treated with PUVA. Methods We prospectively identified cases of melanoma and documented the extent of exposure to PUVA among 1380 patients with psoriasis who were first treated with PUVA in 1975 or 1976. Using incidence data, we calculated the expected incidence of melanoma in this cohort and compared it with the observed incidence. Using regression models, we assessed the risks of melanoma associated with a long time (>15 years) since the first treatment and with a large number of PUVA treatments (>250). Results From 1975 through 1990, we detected four malignant melanomas, about the number expected in the overall population (relative risk, 1.1). From 1991 through 1996, we detected seven malignant melanomas (relative risk, 5.4; 95 percent confidence interval, 2.2 to 11.1). The risk of melanoma was higher in the later period than in the earlier one (incidence-rate ratio, 3.8) and higher among patients who received at least 250 PUVA treatments than among those who received fewer treatments (incidence-rate ratio, 3.1). Conclusions About 15 years after the first treatment with PUVA, the risk of malignant melanoma increases, especially among patients who receive 250 treatments or more.

Stern RS, Liebman EJ, Väkevä L. (1998) "Oral psoralen and ultraviolet-A light (PUVA) treatment
of psoriasis and persistent risk of nonmelanoma skin cancer. PUVA Follow-up Study." J Natl
Cancer Inst. 90(17), 1278-84. Abstract. BACKGROUND/METHODS: The treatment of psoriasis
with high-dose exposure to oral psoralen and ultraviolet-A light (i.e., PUVA) substantially
increases the risk of cutaneous squamous cell cancer, but not of basal cell cancer, within a
decade of beginning treatment. To assess the persistence of cancer risk among individuals
treated with PUVA, including those who discontinued therapy long ago and those without
substantial exposure to other carcinogens, we prospectively studied a cohort of 1380 patients
with psoriasis who were first treated during the period from January 1, 1975, through October 1, 1976, and evaluated risk factors associated with the development of cutaneous squamous cell cancers and basal cell cancers after 1985. RESULTS: From 1975 through 1996, 237 patients
developed 1422 cutaneous squamous cell cancers. From 1986 through 1996, 135 (12.5%) of
1081 patients without a prior squamous cell cancer developed 593 such tumors. From 1975
through 1997, 247 patients developed 1042 basal cell cancers; these patients included 151
individuals with a first basal cell cancer after 1985. Among those without a squamous cell or a
basal cell cancer in the first decade of the prospective study, a strong dose-related increase in
the risk of squamous cell cancer was observed in the subsequent decade (adjusted relative risk
[> or =337 treatments versus <100 treatments] = 8.6; 95% confidence interval = 4.9-15.2). Risk of basal cell cancer was substantially increased only in those patients exposed to very high levels of PUVA (> or =337 treatments). CONCLUSIONS: High-dose exposure to PUVA is associated with a persistent, dose-related increase in the risk of squamous cell cancer, even among patients lacking substantial exposure to other carcinogens and among patients without substantial recent exposure to PUVA. Exposure to PUVA has far less effect on the risk of basal cell cancer. The use of PUVA for psoriasis should be weighed against the increased cancer risk.

Stern R.S. & Lunder E.J. (1998) "Risk of Squamous Cell Carcinoma and Methoxsalen (Psoralen)
and UV-A Radiation (PUVA)." Arch Dermatol 134, 1582-1585. Abstract. Objective To assess the
risk of squamous cell carcinoma (SCC) and the relation of dose to risk among groups of patients
with psoriasis exposed to psoralen–UV-A (PUVA).Data Sources Four electronic databases were
searched from 1984 to 1998.Study Selection In addition to the PUVA Follow-up Study, we
included all English-language studies from the United States and Europe with at least 150
patients enrolled, who were followed up for at least 5 years as identified from our bibliographic
search.Data Extraction A custom-designed questionnaire was used to extract data from each of
the articles. For each study, if possible, we determined the incidence of basal cell carcinomas and SCCs and the incidence rate ratio of SCC among patients exposed to low-dose (we defined as <100 treatments or 1000 J/cm2) compared with high-dose PUVA (>200 treatments or 2000
J/cm2). Exact methods were used to calculate the incidence rate ratios.Data Synthesis In
addition to our study, we identified and reviewed 8 other studies. Overall, the incidence among
patients exposed to high-dose PUVA was 14-fold higher than among patients with low-dose
exposure (95% confidence interval, 8.3-24.1); a greater dose-dependent increase in risk than that observed in the PUVA Follow-up Study. Conclusion Although the incidence of SCC reported
among groups of PUVA-treated patients followed up for at least 5 years varies greatly, compared with the risk in low-dose patients, long-term high-dose exposure to PUVA was consistently observed to significantly increase the risk of SCC in all studies reviewed.

Stern R.S. (2001) "The risk of melanoma in association with long-term exposure to PUVA" J. Am.
Acad. Dermatol 44 (5), 755-761 Abstract Background: Oral methoxsalen (psoralen) and
ultraviolet A radiation (PUVA) is a highly effective therapy for psoriasis and many other skin
conditions. It is carcinogenic. Previously we reported an increased risk of melanoma that first
emerged 15 years after first treatment. Objective: Our purpose is to present additional data
concerning the associations of previous exposure to PUVA, the passage of time and the risk of
malignant melanoma, Methods: We have prospectively studied a cohort of 1380 patients first
treated with PUVA in 1975 and 1976. We have documented the occurrence of melanoma and in
this report compare the observed and expected incidence of melanoma in this cohort, particularly melanomas developing since our earlier report (ie, after March 1996). Results: Since 1975, 23 patients have developed 26 invasive or in situ cutaneous melanomas. In an average of 2.25 years since our last report, we detected 7 additional invasive melanomas (incidence rate ratio, 8.4; 95% confidence interval, 3.4-17.3). Conclusion: Beginning 15 years after first exposure to PUVA, an increased risk of melanoma is observed in our cohort of PUVA-treated patients. This risk is greater in patients exposed to high doses of PUVA, appears to he increasing with the passage of time, and should he considered in determining the risks and benefits of this therapy. Lim JL, Stern RS. (2005) "High levels of ultraviolet B exposure increase the risk of non-melanoma skin cancer in psoralen and ultraviolet A-treated patients." J Invest Dermatol. 124(3), 505-13.  Abstract. Sunlight and psoralen and ultraviolet A (PUVA) are risk factors for the development of squamous cell carcinoma (SCC) and, to a lesser extent, basal cell carcinoma (BCC). Ultraviolet B (UVB) therapy, used for the treatment of psoriasis, might also increase the risk of these tumors. We studied the relation of skin cancer incidence to UVB use among 1380 adult subjects enrolled in a long-term safety trial of PUVA therapy. We used negative binomial regression models to quantify the association between UVB and the development of non-melanoma skin cancer (NMSC), controlling for known confounders. High UVB exposure (> or =300 treatments vs <300 treatments) was associated with a modest but significant increase in SCC (adjusted incidence rate ratio (IRR)=1.37, 95% confidence interval (CI)=1.03-1.83) and BCC (adjusted IRR=1.45, 95% CI=1.07-1.96) risk. Among patients with <100 PUVA treatments, high UVB exposure was significantly associated with the development of SCC (adjusted IRR=2.75, 95% CI=1.11-6.84) and BCC (adjusted IRR=3.00, 95% CI=1.30-6.91) on body sites typically exposed to UVB therapy but not on chronically sun-exposed sites typically covered during therapy. For adults with high UVB exposure levels, UVB confers a modest increase in NMSC risk, much less than that observed with PUVA. Therefore, UVB remains a relatively low-risk treatment for psoriasis.

Tanenbaum L., Parrish J.A., Pathak M.A., Anderson R.B. & Fitzpatrick T.B. (1975) "Tar
photoxicity and phototherapy for psoriasis." Arch Dermatology 111(4) Abstract. The photoxicity of coal tars was determined by comparing the ultraviolet light (UVL) energy required to produce
erythema at tar treated sites (minimal phototoxic dose [MPD]) with the energy required to produce the same degree of erythema at untreated control sites (minimal erythema dose [MED]). The ratio of MED/MPD is the photoxic index (PI). Tars that were phototoxic had a PI of greater than 1. Using a UVA (320 to 400 nm) and a tuvb (290 to 320 nm) light source, 15 subjects and six tars were tested. All tars were phototoxic to UVA but not to UVB (P smaller than 0.0001). Although tar and UVL is a widely accepted treatment for psoriasis (Goeckerman therapy), the light sources employed at normal exposure times provide insufficient UVA energy to produce a phototoxic reaction to the tars are used. The therapeutic response seen in psoriatic patients treated with tar and UVL should therefore not be attributed to tar phototoxicity.

Tanenbaum L., Parrish J.A., Haynes H.A., Fitzpatrick T.B. & Pathak M.A. (1976) "Prolonged
ultraviolet light-induced erythema and the cutaneous carcinoma phenotype." J Invest Dermatol.
67 (4):513-7 Abstract. A considerable amount of evidence exists in support of the role of
ultraviolet radiation as a major etiologic factor in human skin cancer, both melanoma and
carcinoma types. On the basis of epidemiologic studies a phenotype has been described which
helps to identify the persons who are more susceptible to skin cancer. In an attempt to further
define this population, patients with cutaneous carcinoma and a normal control group were
exposed to artificial ultraviolet light (UVL) and the erythema and tanning responses of each group were measured over a 21-day period. UVL-induced erythema was prolonged in a significantly higher percentage of patients with skin cancer than in control patients, lasting two to three weeks after single exposures to 6 and 8 times the patient's minimal erythema dose. The presence of prolonged erythema correlated with this history of previous skin cancer did not correlate with other established risk factors for cutaneous carcinoma, i.e., fair skin, light hair and light eyes, easy sunburning and poor tanning, and Celtic ancestry. Prolonged erythema following UVL radiation may therefore represent an additional risk factor and help to identify the skin cancersusceptible population.

Viola G., Fortunato E., Cecconet L., Del Giudice L., Dall'Acqua F. & Basso G.. (2008) "Central
role of mitochondria and p53 in PUVA-induced apoptosis in human keratinocytes cell line NCTC-
2544." Toxicol Appl Pharmacol. 227(1), 84-96. Abstract. Despite strong evidence concerning the
high efficiency of PUVA therapy (psoralen plus UVA light), its mechanism of action has not yet
been fully elucidated. In this study, we have evaluated in a cell line of human keratinocytes
(NCTC-2544) the effects of two linear psoralen derivatives, 8-methoxypsoralen (8-MOP) and 5-
methoxypsoralen (5-MOP), that are widely used in PUVA therapy and two angular derivatives,
Angelicin (ANG) and 4,6,4'-trymetyl angelicin (TMA). All derivatives photoinduce cellular death,
TMA being the most active compound. The cell cycle analysis showed that the four derivatives
induce, 24 h after irradiation, a cell cycle arrest in G1 phase later followed by massive apoptosis.
The G1 arrest is correlated to an increase in the expression of p21(Waf1/Cip1), a protein
associated with the cell cycle block and apoptosis. Furthermore, treatment of NCTC-2544
resulted in p53 activation by 5-MOP, 8-MOP, and ANG but not TMA and its phosphorylation at
serine-15. The levels of p21(Waf1/Cip1) paralleled p53 protein staining pattern suggesting that
p53 activation correlated with p21(Waf1/Cip1) induction. Simultaneous to p53 activation,
psoralens induced mitochondrial depolarization, cytochrome c release, mitochondrial production
of reactive oxygen species, as well as caspase-3 and -9 activation. Thus these results strongly
indicate the necessity of p53 activation and the induction of the apoptotic machinery downstream of mitochondria.

Reactive Oxygen Species.
Exposure of the skin to UV radiation can result in a number of harmful effects including the generation of reactive oxygen species (ROS). Cellular anti-oxidants can play a part in reducing the occurrence & intensity of photo-aging & UVinduced skin diseases.
Dalle Carbonare M, Pathak MA. (1992) "Skin photosensitizing agents and the role of reactive
oxygen species in photoaging." J Photochem Photobiol B. 14(1-2), 105-24. Abstract. In this
paper, the role of reactive oxygen species in photoaging is presented. Many photosensitizing
agents are known to generate reactive oxygen species (singlet oxygen (1O2), superoxide anion
(O2.-) and .OH radicals). Although photoaging (dermatoheliosis) of human skin is caused by UVB
and UVA radiation, the hypothesis tested here in the pathogenesis of photoaging of human skin is the free radical theory involving the generation of reactive oxygen species by UVA (320-400 nm) radiation and their damaging oxidative effects on cutaneous collagen and other model proteins.
The UVA-generated reactive oxygen species cause cross-linking of proteins (e.g. collagen),
oxidation of sulfydryl groups causing disulfide cross-links, oxidative inactivation of certain
enzymes causing functional impairment of cells (fibroblasts, keratinocytes, melanocytes,
Langerhans cells) and liberation of proteases, collagenase and elastase. The skin-damaging
effects of UVA appear to result from type II, oxygen-mediated photodynamic reactions in which
UVA or near-UV radiation in the presence of certain photosensitizing chromophores (e.g.,
riboflavin, porphyrins, nicotinamide adenine dinucleotide phosphate (NADPH), etc.) leads to the
formation of reactive oxygen species (1O2, O2.-, .OH). Four specific observations are presented
to illustrate the concept: (1) the production of 1O2 and O2.- by UVB, UVA and UVA plus
photosensitizing agents (such as riboflavin, porphyrin and 3-carbethoxypsoralens) as a function
of UV exposure dose, the sensitizer concentration and the pH of the irradiated solution; (2) the
formation of protein cross-links in collagen, catalase and superoxide dismutase by 1O2 and O2.-
(.OH) and the resulting denaturation of proteins and enzyme activities as a function of UVA
exposure dose; (3) the protective role of selective quenchers of 1O2 and O2.- (e.g. alphatocopherol acetate, beta-carotene, sodium azide, ascorbic acid, etc.) against the
photoinactivation of enzymes and the prevention of the protein cross-linking reaction; (4) the
possible usefulness of certain antioxidants or quenchers that interact with the UVA-induced
generation of reactive oxygen species in the amelioration of the process of photoaging.

Hakozaki T., Date A., Yoshii T., Toyokuni S., Yasui H. & Sakurai H. (2007) “Visualization and
characterization of UVB-induced reactive oxygen species in a human skin equivalent model. Arch
Dermatol Res. 2007 Oct 30. Abstract. Reactive oxygen species (ROS) play important roles in the
process of ultraviolet-induced skin damage or photoaging. Although many enzymatic and
chemical methods have been developed for evaluating ROS, evaluation methods for ROS
generation in living systems are quite limited. Here we propose a unique system to visualize
UVB-induced ROS and investigate the biological impact of ROS. In brief, a human skin equivalent
model (HSEM) was exposed to UVB. Emitted luminescence from the HSEM was visualized and
semi-quantified by using a chemiluminescent probe (CLA) and an ultra low-light imaging
apparatus. The effects of anti-oxidative compounds such as ascorbate, beta-carotene,
superoxide dismutase (SOD), and yeast ferment filtrate (YFF) on the HSEM were evaluated by
semi-quantification of emitted chemiluminescence (CL) intensities, MTT assay and 8-hydroxy-2'-
deoxyguanosine (8-OHdG) staining. Visualization of time- and space-dependent dynamics of
ROS generation in the HSEM was successfully achieved by utilizing a sensitive two-dimensional
ultra-low light luminograph. Treatments with beta-carotene and SOD effectively suppressed CL
intensity, indicating the generation of (1)O(2) and O(2) (.) (-) in the HSEM under UVB exposure.
Tested anti-oxidative compounds also attenuated UVB-induced CL and ameliorated the induced
skin damages in terms of 8-OHdG formation and cell death. As a conclusion, this model is useful
for not only visualizing the production of UVB-induced ROS in real-time but also evaluating the
efficacy of topically applied anti-oxidative compounds to suppress ROS generation and attenuate sequential chemical and biological responses.

Pathak M.A. & Joshi P.C. (1984) "Production of active oxygen species (1O2 and O2-.) by
psoralens and ultraviolet radiation (320-400 nm)." Biochim Biophys Acta. 798(1), 115-26.
Abstract. Furocoumarins (psoralens) are potent skin photosensitizing agents that are used in
combination with long-wavelength ultraviolet radiation (320-400 nm) in the treatment of psoriasis and other skin diseases. Twelve linear and angular psoralens, capable of forming monofunctional and bifunctional adducts with DNA, were examined with a view to elucidate the role of 1O2 and O2-. in evoking skin photosensitization reactions and skin carcinogenesis. The results showed that both linear psoralens (capable of forming interstrand cross-links) and isopsoralens (angular, monofunctional type) and 3-carbethoxypsoralen (a linear and monofunctional type) produced 1O2 and O2-., although at varying degrees. Psoralen and 3-carbethoxypsoralen produced 1O2 greater than isopsoralens (angelicins). However, nonphotosensitizing angelicin, 5-methylangelicin, and 4,8-dimethyl-5'-carboxypsoralen produced 1O2 greater than 8-methoxypsoralen and 5- methoxypsoralen. The three monofunctional angelicin derivatives (isopsoralens) produced more O2-. than 8-methoxypsoralen, 5-methoxypsoralen, and 3,4'-dimethyl-8-methoxypsoralen. 3-Carbethoxypsoralen, a potent generator of 1O2 and a moderate producer of O2-., was highly photolabile. Until recently, skin photosensitization reactions (erythema, edema, damage to DNA or the membrane of cutaneous cells, the inhibition of scheduled DNA synthesis and skin carcinogenesis, etc.) were believed to involve photocyclo-addition of psoralens to DNA mediated by a type-I or anoxic reaction (a sensitizer-substrate interaction through the transfer of hydrogen atoms or electrons, but no direct involvement of molecular oxygen). Oxygen-dependent sensitized photodynamic reactions of type-II, involving the production of reactive oxygen (1O2 and O2-.), were believed not to mediate psoralen photosensitization reactions. We suggest that1O2 and O2-. may also articipate in skin photosensitization and cell membrane-damaging reactions. The fact that certain monofunctional isopsoralens produce 1O2 and O2-. at rates comparable to or better than bifunctional psoralens suggests that these reactive moieties of oxygen could play a major role in explaining their recently observed carcinogenic property and cell membrane-damaging reactions (e.g., edema or inflammation, etc.).

Sakurai H, Yasui H, Yamada Y, Nishimura H. & Shigemoto M. (2005) "Detection of reactive
oxygen species in the skin of live mice and rats exposed to UVA light: a research review on
chemiluminescence and trials for UVA protection." Photochem Photobiol Sci. 4(9), 715-20.
Abstract. The harmful effects of ultraviolet (UV) exposure on the skin are associated with the
generation of reactive oxygen species (ROS) such as superoxide anion radical ( O(2)(-)),
hydrogen peroxide (H(2)O(2)), hydroxyl radical ( OH), and singlet oxygen ((1)O(2)) as well as with lipid peroxides and their radicals (LOOH and LOO ). To give direct proof that such ROS are
generated in UV-exposed skin, we proposed the in vivo detection and imaging method in which
both a sensitive and specific chemiluminescence (CL) probe, such as CLA, and an ultralow-light
imaging apparatus with a CCD camera were used. With this method we found that O(2)(-) is
formed intrinsically and that (1)O(2) and O(2)(-) are generated in the UVA-exposed skin of mice.
In addition, we indicated that antioxidative ability against ROS in the skin of hairless rats
decreased as age increased. Using these findings, we demonstrated the protective abilities of
sodium ascorbate, caffeic acid, essential aroma oils, and zinc(ii) ion and its complexes, which we
administered to mice both topically and orally. We present a review for the current state of our
research proposing the sensitive CL method as a useful in vivo tool in photobiological research
for the detection of oxidative stress as well as for the evaluation of antioxidative agents to the
skin.

Yamada Y., Yasui H. & Sakurai H. (2006) "Suppressive effect of caffeic acid and its derivatives
on the generation of UVA-induced reactive oxygen species in the skin of hairless mice and
pharmacokinetic analysis on organ distribution of caffeic acid in ddY mice." Photochem Photobiol.
82(6), 668-76 Abstract. Caffeic acid (CA) and its analogues such as rosmarinic acid are well
known as antioxidative agents. Exposure to UVA is known to generate reactive oxygen species
(ROS) such as singlet oxygen (1O2) and superoxide anion radical (*O2-) in the skin of animals,
which in turn induces skin photodamage and photoaging. Because CA and its analogues quench
1O2, these compounds were topically applied to the abdominal skin of live hairless mice and
were found to suppress ROS generation upon UVA exposure. Furthermore, the generation of
UVA-induced ROS was also suppressed in the skin of mice that were orally given CA. In order to
understand the mechanism by which CA blocks ROS production in UVA-exposed skin, the
pharmacokinetics of CA upon oral administration to mice was followed and CA was found to
efficiently distribute in the skin. These results suggest that skin damage by UVA-induced ROS
generation is reduced by oral supplementation of CA, which has a scavenging and quenching
activity against ROS.

Removal of furanocoumarins from Essential Oils
Radford T., Olansky O.S. (Coca-cola Co.) “Process for Dewaxing Citrus Oils” US Patent Applicn
No 83929 (filed on 1993-06-29).
Cropwatch summary: The patent describes process which claims to convert soralen epoxides to diols which are removed by precipitation or selective dissolution. Other workers have suggested that chilling essential oils will remove some FC content; other methods include alkaline treatment to remove bergapten, fractional distillation to remove some FC content, or various chemio-physical absorption processes. Cropwatch believes that there is no single process will remove 100% of all furanocoumarins without affecting the odour & taste profile of
the raw material.

Reactive oxygen species.
Bakkali F., Averbeck S., Averbeck D., Zhiri A., Baudoux D. & Idaomar M. (2006) "Antigenotoxic
effects of three essential oils in diploid yeast (Saccharomyces cerevisiae) after treatments with
UVC radiation, 8-MOP plus UVA and MMS." Mutat Res. 14;606(1-2),:27-38. Abstract. Essential
oils (EOs) extracted from medicinal plants such as Origanum compactum, Artemisia herba alba
and Cinnamomum camphora are known for their beneficial effects in humans. The present study
was undertaken to investigate their possible antigenotoxic effects in an eukaryotic cell system,
the yeast Saccharomyces cerevisiae. The EOs alone showed some cytotoxicity and cytoplasmic
petite mutations, i.e. mitochondrial damage, but they were unable to induce nuclear genetic
events. In combination with exposures to nuclear mutagens such as 254-nm UVC radiation, 8-
methoxypsoralen (8-MOP) plus UVA radiation and methylmethane sulfonate (MMS), treatments
with these EOs produced a striking increase in the amount of cytoplasmic petite mutations but
caused a significant reduction in revertants and mitotic gene convertants induced among
survivors of the diploid tester strain D7. In a corresponding rho0 strain, the level of nuclear
genetic events induced by the nuclear mutagens UVC and 8-MOP plus UVA resulted in the same
reduced level as the combined treatments with the EOs. This clearly suggests a close
relationship between the enhancement of cytoplasmic petites (mitochondrial damage) in the
presence of the EOs and the reduction of nuclear genetic events induced by UVC or 8-MOP plus
UVA. After MMS plus EO treatment, induction of these latter events was comparable at least per
surviving fraction in wildtype and rho0 cells, and apparently less dependent on cytoplasmic petite induction. Combined treatments with MMS and EOs clearly triggered switching towards late apoptosis/necrosis indicating an involvement of this phenomenon in EO-induced cell killing and concomitant decreases in nuclear genetic events. After UVC and 8-MOP plus UVA plus EO
treatments, little apoptosis and necrosis were observed. The antigenotoxic effects of the Eos
appeared to be predominantly linked to the induction of mitochondrial dysfunction.

Sakurai H., Yasui H., Yamada Y., Nishimura H. |& Shigemoto M. (2005) "Detection of reactive
oxygen species in the skin of live mice and rats exposed to UVA light: a research review on
chemiluminescence and trials for UVA protection." Photochem Photobiol Sci. 4(9), 715-20
Abstract. The harmful effects of ultraviolet (UV) exposure on the skin are associated with the
generation of reactive oxygen species (ROS) such as superoxide anion radical (O(2)(-)),
hydrogen peroxide (H(2)O(2)), hydroxyl radical ( OH), and singlet oxygen ((1)O(2)) as well as with lipid peroxides and their radicals (LOOH and LOO ). To give direct proof that such ROS are
generated in UV-exposed skin, we proposed the in vivo detection and imaging method in which
both a sensitive and specific chemiluminescence (CL) probe, such as CLA, and an ultralow-light
imaging apparatus with a CCD camera were used. With this method we found that O(2)(-) is
formed intrinsically and that (1)O(2) and O(2)(-) are generated in the UVA-exposed skin of mice.
In addition, we indicated that antioxidative ability against ROS in the skin of hairless rats
decreased as age increased. Using these findings, we demonstrated the protective abilities of
sodium ascorbate, caffeic acid, essential aroma oils, and zinc(ii) ion and its complexes, which we
administered to mice both topically and orally. We present a review for the current state of our
research proposing the sensitive CL method as a useful in vivo tool in photobiological research
for the detection of oxidative stress as well as for the evaluation of antioxidative agents to the
skin.

Repair of cellular furanocoumarin-UVA damage / UVA-damage
Averbeck D, Averbeck S. (1998) "DNA photodamage, repair, gene induction and genotoxicity
following exposures to 254 nm UV and 8-methoxypsoralen plus UVA in a eukaryotic cell system."
Photochem Photobiol. 68(3):289-95. Abstract. The induction and repair of different types of
photodamage and photogenotoxicity in eukaryotic cells have been the subject of many studies.
Little is known about possible links between these phenomena and the induction of DNA
damage-inducible genes. We explored this relationship using the yeast Saccharomyces
cerevisiae, a pertinent eukaryotic model. Previous results showed that the photogenotoxic
potential of 8-methoxypsoralen (8-MOP) plus UVA is higher than that of UV (254 nm). Moreover,
the induction of the ribonucleotide reductase gene RNR2 by UV and 8-MOP plus UVA in an
RNR2-LACZ fusion strain and the formation of DNA double-strand breaks (dsb) as repair
intermediates after such treatments suggest that the latter process could involve a signal for gene induction. To further substantiate this, we measured the induction of the DNA repair gene RAD51 in RAD51-LACZ fusion strains using the dsb repair and recombination deficient mutant rad52 and the corresponding wild type, and we determined the formation of dsb by pulsed-field gel electrophoresis. After treatments, the resealing of dsb formed as repair intermediates was
impaired in the rad52 mutant. At equal doses, i.e. the same number of lesions, the induction of
the RAD51 gene by UV or 8-MOP plus UVA was significantly reduced in the rad52 mutant as
compared with the wild type. The same was true when equitoxic doses were used. Thus, the
RAD52 repair pathway appears to play an important role not only in dsb repair but also in gene
induction. Furthermore, the signaling pathways initiated by DNA damage and its processing are
somewhat linked to the photogenotoxic response.

Dardalhon M. & Averbeck D. (1995) "Pulsed-field gel electrophoresis analysis of the repair of
psoralen plus UVA induced DNA photoadducts in Saccharomyces cerevisiae." Mutat Res.
336(1):49-60. Abstract. In the yeast Saccharomyces cerevisiae, double-strand breaks (DSB)
have been observed during the DNA repair of psoralen plus UVA induced lesions. In the present
paper, we analyzed this repair step in some detail using pulsed-field gel electrophoresis (CHEF)
to get a better understanding of this phenomenon with regard to the type of lesions induced and the repair pathways involved. The results confirm that, during post-treatment incubation of
Saccharomyces cerevisiae cells, DSB are formed. Their appearance is dose-dependent and the
rate of induction is comparable in large (chromosome IV) and small (chromosome III)
chromosomes. The formation of DSB is evidenced by the breakage of linear chromosomes III and IV, but also, after high doses, by the linearization of a circular form of chromosome III. The
induction of DSB appears to be highly dependent on the induction of interstrand cross-links since they are clearly present after treatments with 8-MOP plus 365 nm radiation (inducing
monoadducts and cross-linking in DNA), but practically absent after treatment with 8-MOP plus
405 nm radiation (inducing predominantly monoadducts) at comparable levels of photoadducts.
The occurrence of DSB is dependent on the RAD2 and RAD52, but not on the RAD6 gene. It is
likely that the specific processing of DNA lesions involving DSB is related to the genotoxic
consequences observed.

Webb R.B. & Lorenz J.R. (1970) "Oxygen dependence & repair of lethal effects of near ultraviolet
& visible light." Photochemistry and Photobiology 12(4), 283–289. Abstract. Lethality in a
repairable strain (WP2) and an excision repair deficient strain (WP2hcr) of Escherichia coli was
studied at wavelengths of 254, 313, 365, and 390–750 nm. Survival curves were empirically fitted to the expression S= 1 - (1-e-kl)", where S is the fraction surviving, D is the incident dose in ergs mm-2, k is the inactivation constant in units of (erg mm-2)-1 and n is the ‘shoulder constant’. The repairable sector (k(hcr-)–k(hcr-)lk(hcr-), a conservative estimate of the repair capability of E. coli WP2, was 0.91 at 254 nm, 0.92 at 313 nm, 0.60 at 365 nm, and 0.13 at 390–750 nm. Although there was no oxygen enhancement of inactivation at 254 nm and 313 nm, a strong enhancement was identified at 365 nm and 390–750 nm. These results suggest that oxygen-dependent damage induced by near u.v. (365 nm) can be partially repaired by the excision-repair system in E. coli.

Rue oil Ruta graveolens L.
CAS n°: 8014-29-7; EINECS CAS n° 84929-47-5
Cropwatch summary:
Phototoxicity: IFRA limits rue oil to 0.15% in fragrances not washed off the skin exposed to sunshine. Eickhorst et al (2007) describe photo-irritancy as the reaction to human skin that has been in contact with rue herb & exposed to UV light.
Composition: Jaacob (1989) through Joulain (2006) steam distilled Malaysian rue oil contains psoralen (1.3%) and bergapten (7.2%). Although rue oil is not much used in perfumery, RIFM give the following data “measured by the fragrance industry” (??) for Rue oil (but no botanical or geographical source details given): psoralen 150ppm, bergapten 150 ppm, xanthotoxin 320 ppm, isopimpernellin 200 ppm, angelicin 430 ppm; bergamottin not detected (RIFM Fact Sheet No 3.).

Eickhorst K, DeLeo V, Csaposs J. (2007) “Rue the herb: Ruta graveolens - associated
phytophototoxicity.” Dermatitis 18(1), 52-5. Abstract. We describe an unusual case of
phytophototoxicity induced by an herbal plant, Ruta graveolens, from the Rutaceae family. This
common herb, also called rue, can be found throughout rural settings in the United States. When psoralens from rue come in contact with human skin that is subsequently exposed to ultraviolet A light, an impressive photoirritant reaction can occur. This report both clarifies the distinguishing features of photoirritant reactions versus photoallergic reactions and reviews the pathophysiology and clinical presentation of phytophotodermatitis. R. graveolens can be associated with an impressive photoirritant reaction and should not be used as an insect repellent.

Schimmer O, Kühne I. (1990) "Mutagenic compounds in an extract from Rutae Herba (Ruta
graveolens L.). II. UV-A mediated mutagenicity in the green alga Chlamydomonas reinhardtii by
furoquinoline alkaloids and furocoumarins present in a commercial tincture from Rutae Herba."
Mutat Res. 243(1):57-62. Abstract. A commercial tincture prepared from Rutae Herba (Ruta
graveolens L.) exhibited a moderate photomutagenicity in an arginine-requiring mutant strain of
Chlamydomonas reinhardtii. In the tincture some furocoumarins, e.g., bergapten, psoralen,
imperatorin, and 3 furoquinoline alkaloids (dictamnine, gamma-fagarine, skimmianine) were
detected. All compounds revealed photomutagenic properties but their activities were quite
different. Bergapten was the most potent furocoumarin. Dictamnine, the furoquinoline with the
strongest effect, reached only about 10% of the activity of bergapten. Based on the amount of
these compounds in the tincture and their activities we conclude that bergapten is mainly
responsible for the photomutagenicity of the tincture. The lower phototoxicity and
photomutagenicity of the furoquinoline alkaloids may be due to the fact that furoquinolines form
only monoadducts with DNA in the presence of UV-A in contrast to furocoumarins which also
form biadducts.

Satsuma oil (Japan, USA) Citrus nobilis var. unshiu Marc.
Cropwatch summary: No data.

SCC(NF)P opinions on furanocoumarins.
SCCNFP/0392/00 Opinion 25th Sept 2001, SCCP proposed to permit the use of furocoumarin-containing essential oils as long as the total concentration of “furocoumarins and furocoumarin-like substances” in the finished cosmetic product does not exceed 1 ppm. Cropwatch comments: This Opinion has been widely criticised, as there is no understood definition of ‘furocumarin-like
substances’, no available analytical method to determine all furocoumarins & ‘furocoumarin-like substances’, and anyway, not all furocoumarins show adverse photo-toxic effects.

SCCNFP/0740/03 Opinion 20th Oct 2003 on bergamottin concluded that  Photomutagenicity
and photo-carcinogenicity are the main effects of concern in relation to the use of furocoumarins in cosmetics The data submitted by EFFA on bergamottin is not adequate for evaluation of the safety of the substance in relation to photomutagenicity and photocarcinogenicity.”

SCCP/0942/05 Opinion 13th Dec 2003 concluded that “The data provided do not justify a higher limit than 1ppm (not intentionally added) for furocoumarins in cosmetics.” Cropwatch comments: Cropwatch immediately rushed out a article spelling out the devastating impact this restriction would have on the fragrance trade at http://www.cropwatch.org/newslet3.htm. The SCCP Opinion is, of course, unworkable since no method exists for determining FC concentrations in aroma ingredients, and formulators do not have the information on FC
concentrations in aroma ingredients to hand. In any case not all FC’s show
photo-toxic effects.

SCCNFP/0765/03 Opinion 9th Dec 2003 on sun protection & bronzing products.
The SCCP concluded that “the data available from in vitro short-term studies, experimental studies in animals, and epidemiological studies on humans on the effect of furocoumarines in sun-protection and sun bronzing product do not justify a higher limit than 1 ppm (not to be intentionally added) for furocoumarines (sic) in cosmetics. This Opinion should be interpreted in conjunction with the Opinion on CMR substances (SCCNFP/0474/01, final, Adopted 25 September 2001).”
Cropwatch comments: a curiously narrow band of available evidence was considered and the judgements made appear to be politically motivated, rather than scientific. Again, the Opinion is unworkable in practice for the same reasons cited above.

SCCNFP/0761/03 Opinion 9th Dec 2003 on photomutagenicity of isopimpinellin
in cosmetic products. Cropwatch comments: The SCCP decided that ‘submitted data’ - seemingly from an unpublished report to RIFM by Ballantyne M. (2003) - demonstrated that isopimpinellin is photomutagenic in Salmonella typhimurium TA102, but that “there is incomplete information on photomutagenicity and on photoclastogenicity of isopimpinellin to enable a safety evaluation…”

Ballantyne M. (2003) “Isopimpinellin: Reverse mutation in five histidine-requiring strains of
Salmonella typhimurium, in the presence of ultra violet light.” Research Institute for Fragrance
Materials, Inc., 2003.

Skimmea laureola oil India, Kasmir Skimmia laureola Sieb. et. Zucc. ex Walp
Cropwatch summary: steam distilling leaves yield oil produced on commercial scale, which has been compared as similar in odour to petitgrain. 25 psorelens & coumarins amongst identified FC’s (Pathak & Pant 1977; Radzan et al. 1986).
Acetone extract of dried leaves yields bergatene, xanthotoxin etc. (Bhargava et
al. 1973).

Star anise Oil, China Illicium verum Hook f.
CAS n° 84650-59-9; EINECS-CAS n°: 84650-59-9
Cropwatch summary: Not previously found phototoxic to mice & swine (Opdyke
1975).

Sweetie oil Citrus paradisi Osbeck x Citrus grandis Mcfad.
Cropwatch summary: No data. Saita et al. (2004) screened the FC content of
fifteen citrus fruits by enzyme-linked immunosorbent assay, finding strong
positive reactions in eight of them, including sweetie fruits.

Saita T., Fujito H. & Mori M. (2004) "Screening of furanocoumarin derivatives in citrus fruits by
enzyme-linked immunosorbent assay" Biol. Pharm. Bull. 27(7), 974-977.

Tagetes oil. Mainly from Tagetes minuta but possibly other Tagetes spp. such T.
erecta.

CAS n°: 8016-84-0; EINECS-CAS n°: 91770-75-1
Cropwatch summary:
Phototoxicity: Virtually no history of adverse effects of Tagetes qualities in use, but some photo-toxicity ascribed to contained thiophene compounds, especially a-tertthienyl (Cropwatch 2005). Cropwatch has found a producer interested in a collaboration to produce Tagetes qualities with zero content of thiophenes, thus hopefully circumventing toxicological intervention Meanwhile RIFM have issued test results for Tagetes qualities using the EpiDerm test model in the presence & absence of UV light. Results indicated a potential for phototoxic effects for Tagetes minuta absolute (South Africa) and Tagetes patula absolute (Egypt), but no phototoxic potential for Tagetes minuta oil (Egypt) and Tagetes minuta oil (South Africa). REXPAN decidede that the in vitro tests were not robust enough to determine any NOEL’s, and subsequently RIFM have contracted further studies out to two private laboratories for in vivo work on animals (hairless mice), due to be published in April 2008. No psoralen content was found in any of the samples in contrast to the RIFM information previously given for Tagetes oil (see immediately below).
Composition: RIFM give the following data “measured by the fragrance industry” (??) for tagetes oil (but no botanical or geographical source details given): psoralen 110 ppm, bergapten, xanthotoxin, isopimpinellin, bergamottin & angelicin not detected (RIFM Fact Sheet No 3.).

Tangelo oil – cold pressed.
Cropwatch summary:
FC’s not present in cold-pressed oil according to Widner
(2005).
Widner M. 2005 “Tangerine/grapefruit Hybrids (Tangelos) do not have Furanocoumarins
Associated with Grapefruit/Drug interactions” J Food Sci. 70(4), 307-312.

Tangerine oil cold-pressed Citrus reticulata Blanco var. mandarin
CAS n° 8008-31-9; EINECS-CAS n° 93686-22-7
Cropwatch summary: McHale & Sheridan (1989) found a similar pattern of polymethoxyflavones to sweet orange oil, but with a higher tangeritin content. Cold-pressed tangerine oil contains 50 ppm bergaptene (IFRA). Widmer (2005) found no FC monomer/dimer in 12 tangelos & 2 monohybrid tangerine varieties grown in Florida.

Terpeneless essential oils – furanocoumarins.
Chouchi D. & Barth D. (1994) "Rapid identification of some coumarin derivatives in deterpenated
citrus peel oil by gas chromatography." J Chromatogr A. 672(1-2), 177-83. Abstract. Generally on the gas chromatogram of a volatile essential oil, terpenes, oxygenated compounds and
sesquiterpenes appear. With temperature programming, it was shown that some non-volatiles are present with the volatiles. They are simple coumarin (2H-1-benzopyran-2-one) derivatives such as citropten (5,7-dimethoxycoumarin) and furocoumarins (psoralen, 7H-furo[3,2-g][1]benzopyran- 7-one) such as bergapten (5-methoxypsoralen), some of which are phototoxic.

Terpeneless oils are used in perfumes and cosmetics, so it is important to be able to establish rapidly if they contain phototoxic compounds.

Umbelliferae – furanocoumarin occurrence
Ceska O., Chaudhary S.K., Warrington P.J., Ashwood-Smith M.T. (1987). “Photoactive
furanocoumarins in fruits of some umbellifers.” Phytochemistry 26, 165-169.

Cai Y., Bennett D., Nair R.V., Ceska O., Ashwood-Smith M. J. & DiGiovanni J. (1993) “Inhibition
and Inactivation of Murine Hepatic Ethoxy- and Pentoxyresorufin O-Dealkylase by Naturally
Occurring Coumarins.” Chem. Res. Toxicol. 6, 872-879. Quote: “Humans are also exposed to
furanocoumarins (e.g., bergapten, 6 and xanthotoxin, 7) in umbelliferous vegetables such as
parsnips, celery, and parsley in substantial amounts (ref 4 and references therein). For example, parsnip root reportedly contains as much as 40 mg/kg of certain linear furanocoumarins such as psoralen, xanthotoxin (7), and bergapten (6) which are not destroyed by normal cooking procedures (boiling or microwave) (4).”

UV light.
Ultraviolet spectral band designations recommended by the CIE (Commission Internationale de L'Eclairage) are:
UVA = 315-400nm
UVB = 280-315nm
UVC = 100-280nm
but other designations exist.

Lavker R.M., Gerberick G.F., Veres D., Irwin C.J. & Kaidbey K.H. (1995) "Cumulative effects from
repeated exposures to suberythemal doses of UVB and UVA in human skin." J Am Acad
Dermatol. 32(1), 53-62. Abstract. BACKGROUND: The skin is repeatedly exposed to solar UV
radiation. Long-term photodamage is a consequence of cumulative UV radiation injury. Hence an
examination of the repetitive effects of UV exposure is more likely to yield clues to the early
alterations that lead to photoaged skin than a single exposure. OBJECTIVE: We examined the
effects of repetitive low-dose UV irradiation on human skin with the aim of identifying UVAinduced effects that may have a different wavelength dependence than acute erythema.
METHODS: Areas on the lower part of the back were each exposed to a suberythemal dose (0.5 minimal erythema dose [MED]) of solar simulated radiation (290 to 400 nm) and of UVA (320 to 400 nm) once daily, 5 days a week, for 28 doses. One site was also treated daily with a
sunscreen having a sun protection factor of 22 and then exposed to 11 MEDs of solar simulated
radiation for the same duration. Epidermal and dermal changes were analyzed and quantified by histochemical stains in combination with computer-assisted image analysis of tissue sections.
RESULTS: At equal 0.5 MED doses, UVA induced greater cumulative changes than solar
simulated radiation, as assessed by development of a greater cumulative erythema response in
the first week of treatment, the presence of epidermal hyperplasia and stratum corneum
thickening, depletion of Langerhans cells, dermal inflammatory infiltrates, and deposition of
lysozyme on elastin fibers. These changes were not prevented by the sunscreen. A single shortterm dose of UVA did not elicit these changes. CONCLUSION: These findings suggest that UVA may contribute significantly to long-term actinic damage and that the spectral dependence for cumulative damage does not parallel the action spectrum for acute injury (erythema) in human beings.

Sheehan JM, Cragg N, Chadwick CA, Potten CS, Young AR.(2002) "Repeated ultraviolet
exposure affords the same protection against DNA photodamage and erythema in human skin
types II and IV but is associated with faster DNA repair in skin type IV." J Invest Dermatol.
118(5), 825-9. Abstract. We have investigated the photoprotective properties of induced
pigmentation using erythema and epidermal DNA photodamage as endpoints. Previously
unexposed buttock skin of 12 young, healthy adults (six skin type II and six skin type IV) was
exposed daily (Monday to Friday) for 2 wk (days 1-12) with 0.65 minimal erythema dose of solar
simulated radiation. Mean skin type IV minimal erythema dose was 1.8-fold greater than for skin
type II. Compared to skin type II, solar simulated radiation treatments produced less erythema
and more tanning in skin type IV. To assess DNA photodamage, biopsies were taken and
prepared for paraffin sections that were stained with a monoclonal antibody for thymine dimers.
Thymine dimers were quantified by image analysis. The single exposure data (0.65 and 2
minimal erythema dose) showed that DNA damage was related to physical dose (J per cm2)
independent of skin type. Our data also showed that DNA photodamage accumulates in both skin types with repeated, suberythemal doses of solar simulated radiation. On day 12, there were more thymine dimers in skin type IV than skin type II, again indicating that physical rather than biologic dose determines the level of DNA damage. Comparisons on days 12 and 19, however, showed a much greater loss of thymine dimers in skin type IV, suggesting better thymine dimer repair. Protection factors for erythema and thymine dimers were calculated and shown to be about 2 in both skin types. This provides further indirect evidence that DNA is a chromophore for erythema, but also suggests that a tan may not be the major factor in natural photoprotection.

Tanenbaum L., Parrish J.A., Haynes H.A., Fitzpatrick T.B. & Pathak M.A. (1976) "Prolonged
ultraviolet light-induced erythema and the cutaneous carcinoma phenotype." J Invest Dermatol.
67 (4):513-7 Abstract. A considerable amount of evidence exists in support of the role of
ultraviolet radiation as a major etiologic factor in human skin cancer, both melanoma and
carcinoma types. On the basis of epidemiologic studies a phenotype has been described which
helps to identify the persons who are more susceptible to skin cancer. In an attempt to further
define this population, patients with cutaneous carcinoma and a normal control group were
exposed to artificial ultraviolet light (UVL) and the erythema and tanning responses of each group were measured over a 21-day period. UVL-induced erythema was prolonged in a significantly higher percentage of patients with skin cancer than in control patients, lasting two to three weeks after single exposures to 6 and 8 times the patient's minimal erythema dose. The presence of prolonged erythema correlated with this history of previous skin cancer did not correlate with other established risk factors for cutaneous carcinoma, i.e., fair skin, light hair and light eyes, easy sunburning and poor tanning, and Celtic ancestry. Prolonged erythema following UVL radiation may therefore represent an additional risk factor and help to identify the skin cancersusceptible population.

Verbena (lemon) oil syn Vervein oil. Aloysia triphylla (L'Herit.) Britton syn. Lippia
citriodora (Ort. et Pers.) H.B. & K. syn. Lippia citriodora Kunth.
CAS n° : 8024-12-2 (or 908024-12-2); EINECS-CAS n°: 85116-63-8
Cropwatch summary: Banned IFRA, but RIFM asking industry for technical information (2007). Tisserand & Balacs (1995) assume phototoxicity due to photocitrals, but SCCP hint at FC’s as (additionally?) being responsible. Burfield (2007) previously noted small amount of aromatherapy use apparently without adverse skin reactions. ‘Verbena oil’ as offered commercially however is invariably adulterated or reconstituted. Some commercially produced oil in S. Mediterranean area is obtained by distilling verbena herb tops over lemon oil,
Tisserand R.B. & T. Balacs (1995) Essential oil safety: a guide for healthcare professionals.
Churchill-Livingstone 1995.

Verbena absolute
syn Vervein oil Aloysia triphylla (L'Herit.) Britton syn. Lippia citriodora (Ort. et
Pers.) H.B. & K. syn. Lippia citriodora Kunth.
CAS No: 908024-12-2; EINECS-CAS: 85116-63-8
Cropwatch summary:
Photo-toxicity Opdyke (1992) reported verbena absolute as non-phototoxic. Earlier Forbes & Davies (1980) had reported that verbena absolute applied undiluted to backs of hairless mice followed by UVA radiation produced no phototoxic effects. Insufficient experimental details were provided (e.g. chemical composition, radiation dose) to draw any robust conclusions. Verbena absolute is restricted according to the IFRA website (downloaded 20.03.08) to 0.2% in leave
on products and 2.0% in non-skin contact products, but then confusingly “Verbena absolute obtained from Lippia citriodora Kunth, should not be used as a fragrance ingredient at a level over 1% in fragrance compounds” (but then no indication that the older ‘20% rule’ applies (which, it can only be assumed, does??). And further “this recommendation is based on test results of RIFM showing sensitization reaction at 12% and no sensitization reaction at 2%” (but then how would we know ? - Cropwatch cannot find experimental details within the public domain).

Xanthotoxin (8-Methoxypsoralen; 8-MOP)
The IARC concludes that there is sufficient evidence from epidemiological studies to classify xanthotoxin as carcinogenic to humans (IARC 1987). Some evidence that myristicin increases the photo-toxicity of xanthotoxin in the presence of UV light (Neal 2005).
“Most information on the toxic effects of 5-MOP & 8-MOP comes from studies in patients undergoing treatment for psoriasis and other skin diseases (PUVA therapy) “ (Dept of Health 1996 Report on Toxicity, Muragenicity, Carcinogenicity of Chemicals in Food, Consumer Products & the Environment pub DoH 1998). According to IFRA (2007), xanthotoxin is present at 30ppm max. in rue oil -no botanical or geographic origin cited - & in lime oil (< 5ppm) – type, botanical &
geographic origin not stated, and it is not detected in angelic root oil, bergamot, mandarin, grapefruit & orange oil bitter - no botanical & geographic origins stated.

Attempts for find concentration thresholds for no response effects for xanthotoxin
in PUVA creams have been attempted (Nimkulrat et al. 2005).

Averbeck D, Averbeck S.(1998) "DNA photodamage, repair, gene induction and genotoxicity
following exposures to 254 nm UV and 8-methoxypsoralen plus UVA in a eukaryotic cell system."
Photochem Photobiol. 68(3), 289-95. Abstract. The induction and repair of different types of
photodamage and photogenotoxicity in eukaryotic cells have been the subject of many studies.
Little is known about possible links between these phenomena and the induction of DNA
damage-inducible genes. We explored this relationship using the yeast Saccharomyces
cerevisiae, a pertinent eukaryotic model. Previous results showed that the photogenotoxic
potential of 8-methoxypsoralen (8-MOP) plus UVA is higher than that of UV (254 nm). Moreover,
the induction of the ribonucleotide reductase gene RNR2 by UV and 8-MOP plus UVA in an
RNR2-LACZ fusion strain and the formation of DNA double-strand breaks (dsb) as repair
intermediates after such treatments suggest t