Gielen Aroma

vervolg furocoumarine 1

VERVOLG COUMARINE 1. 

Wen Y.H., Sahi J., Urda E., Kulkarni S., Rose K., Zheng X., Sinclair J.F., Cai H., Strom S.C.,
Kostrubsky V.E. (2002) “Effects of bergamottin on human and monkey drug-metabolizing
enzymes in primary cultured hepatocytes.” Drug Metabolism Disposition 30, 977-984. Abstract.
We investigated the effect of bergamottin, a major furanocoumarin in grapefruit juice, on phase I and phase II drug-metabolizing enzymes using cultured human and monkey hepatocytes. Both
cultured systems were compared and evaluated for the direct effects of bergamottin as well as
control treatments on liver enzymes. Treatment of hepatocytes with 0.1, 1, 5, and 10 microM
bergamottin resulted in a concentration-dependent reduction in CYP3A4 activity (40-100%) in
both human and monkey cells, as measured by testosterone 6 beta-hydroxylase activity.
Bergamottin was potent at eliciting these inhibitory effects at both basal and induced states of
CYP3A. Bergamottin (5 microM) completely inhibited alpha-naphthoflavone-induced
ethoxyresorufin O-dealkylase (EROD) and methoxyresorufin O-dealkylase (MROD) activities in
human hepatocytes and caused a 100% decrease in EROD activity in monkey hepatocytes. A
48-h exposure of cultured human hepatocytes to bergamottin resulted in increased levels of
immunoreactive CYP3A4, CYP1A1, and CYP1A2 proteins, and CYP3A4, CYP1A1, CYP1A2,
CYP2B6, and UDP-glucuronosyl transferase mRNAs. There was only a 20 to 30% reduction in
glucuronidation and sulfation of 4-methylumbelliferone in human hepatocytes by 10 microM
bergamottin and no effect on conjugation in the monkey hepatocytes. These results suggest that bergamottin causes both inhibition of CYP3A and CYP1A1/2 enzymatic activities and induction of correspondent proteins and mRNAs.

Bergapten (5-methoxypsoralen or 5-MOP).
CAS n°: 484-20-8
Classified as probably carcinogenic to humans (IARC 1987). Photo-toxic to umans at 5 ppm (Dubertret et al. 1990). Young et al. (1988) found that a 5-MOP induced tan is protective against the DNA-damaging effects of solar radiation.
Young et al (1990) also found 5-MOP photocarcinogenic in humans.
Papadopoulo & Averbeck (1985) showed 5-MOP & 8-MOP produce genotoxic effects & DNA adducts in Chinese hamster V79 cells.
IFRA bases its Standards/restrictions for bergapten-containing essential oils on the published phototoxic effects of bergapten and the established dose-effect relationships - according to Young et al. (1990) & Dubertret et al. (1990a, 1990b).
Bergapten found at 1671.1 ppm by UV-DAD in cold-pressed bitter orange oil Citrus aurantium L. subsp. amara; at 531.2 ppm by UV-DAD in cold-pressed Israeli grapefruit oil Citrus paradisi Macf., and at 15.69 ppm by UV-DAD in coldpressed Californian lemon oil Citrus limonum (L.) (N. L. Burman) (Frerot & Decorzant 2004).

Averbeck, D., S. Averbeck, L. Dubertret, A. R. Young, P. (1990) “Genotoxicity of bergapten and
bergamot oil in Saccharomyces cerevisiae.” Journal of Photochemistry and Photobiology. B,
Biology 7, 209-229. Abstract. In order to determine the genotoxic potential of bergapten (5-
methoxypsoralen (5-MOP] and bergamot oil (BO), the genetic effects of 5-MOP and BO
(containing equivalent amounts of 5-MOP) were studied in haploid and diploid yeast
(Saccharomyces cerevisiae) using solar simulated radiation (SSR). At equal doses of SSR, equal
concentrations of 5-MOP alone or 5-MOP in BO have a similar influence on survival and on the
induction of cytoplasmic "petite" mutations, reverse and forward mutations, mitotic gene
conversion and genetically aberrant colonies including mitotic crossing over. No reciprocity is
found between SSR dose and 5-MOP concentration for cytotoxic, mutagenic and recombinogenic
effects. In the presence of chemical filters (Parsol 1789, a UVA filter, and Parsol MCX, a
cinnamate derivative acting as a UVB filter) considerable protection is observed against the
induction of genetic effects by 5-MOP and BO containing 5-MOP in haploid and diploid cells. As
indicated by the lower induction kinetics, the protection is higher than expected from the lightabsorbing properties, suggesting photochemical interaction. The protection is slightly higher for BO than for 5-MOP. The induction of genetic effects by 5-MOP alone or BO containing 5-MOP is independent of oxygen. Experiments on suction blister fluids taken from patients after topical
treatment with BO containing 5-MOP indicate that in comparison with water the bioavailability and thus the genotoxic effects of the compounds are decreased. Moreover, in addition to the filtering effect against the photoinduced genotoxic effects of BO, the presence of chemical filters
apparently reduces the penetration of BO containing 5-MOP and provides a reduction in
biological effectiveness.

Dubertret L., Serraf-Tircazes D., Jeanmougin M., Morliere P., Averbeck D., & Young A. R.
(1990a) “Phototoxic properties of perfumes containing bergamot oil on human skin:
Photoprotective effect of UVA and UVB sunscreens.” Journal of Photochemistry and
Photobiology B: Biology 7, 251-259. Abstract. As part of an international cooperative study of the photophysical, photomutagenic and photocarcinogenic properties of bergamot oil and the effect of UVA and UVB sunscreens, the phototoxic properties of model perfumes containing 5, 15 and 50 ppm 5-methoxypsoralen (5-MOP) in bergamot oil with and without a sunscreen have been investigated on human skin. It has been confirmed that the photosensitivity of human skin is maximal 2 h after perfume application. Interestingly the addition of a UVA sunscreen is more
efficient for decreasing the phototoxic properties of bergamot oil than is a UVB sunscreen. The
addition of sunscreens in a model perfume containing 50 ppm 5-MOP in bergamot oil can reduce
the phototoxic properties of this perfume to a toxicity equivalent to that produced by the
application of a model perfume containing 15 ppm 5-MOP without sunscreens. However, despite
their promising protective effect in vitro, UVB and UVA sunscreens at low concentration (0.5%-
1%) in perfumes cannot suppress the phototoxicity of bergamot oil on human skin.
Dubertet et al (1990b) Journal of Photochemistry and Photobiology B: Biology 7, 382.
Frérot E. & Decorzant E. (2004) "Quantification of total furocoumarins in citrus oils by HPLC
coupled with UV, fluorescence and mass detection." J Agr. Food Chem. 52: 6879-6886
IARC (1987) “IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. 5-
Methoxypsoralen and 8-Methoxypsoralen plus ultraviolet irradiation.” Suppl 7, 242-245.
Papadopoulo, D. & Averbeck D. (1985) “Genotoxic effects and DNA photoadducts induced in
Chinese hamster V79 cells by 5-methoxypsoralen and 8-methoxypsoralen.”Mutation
Research/Fundamental and Molecular Mechanisms of Mutagenesis 151, 281-291.
Papadopoulo D. & Moustacchi E. (1990) "Mutagenic effects photoinduced in normal human
lymphoblasts by a monofunctional pyridopsoralen in comparison to 8-methoxypsoralen.” Mutat
Res 245, 259-266.
Walter J.F., Gange R.W. & Mendelson IR. (1982) "Psoralen-containing sunscreen induces
phototoxicity and epidermal ornithine decarboxylase activity." Am Acad Dermatol. 6(6), 1022-7.
Abstract. Sunscreens containing 5-methoxypsoralen (5-MOP) are being promoted commercially
to increase suntanning and sun protection. A recent study indicated that the 5-MOP concentration used in these sunscreens is too low to induce cutaneous phototoxicity with ultraviolet (UV) radiation. We investigated whether the sunscreen Sun System III (SS III), which contains 5-MOP, could induce skin erythema, edema, delayed pigmentation, and epidermal ornithine decarboxylase (ODC) activity when used in conjunction with UVA radiation (320-400 nm). ODC induction is an early event in the promotion of skin tumors. Increased epidermal ODC activity has been reported after exposure to UVB radiation (290-320 nm) alone and with topical 8-methoxypsoralen (8-MOP) plus UVA radiation. Using a solar simulator, we found SS III-induced
erythema, edema, and epidermal ODC activity in hairless mouse skin with only 5 joules/cm2 of
UVA. Human skin showed erythema and delayed pigmentation with SS III plus 20 joules/cm2 of
UVA. No phototoxicity was seen in human skin unless the solar simulator output was filtered
through water to reduce infrared radiation. This indicates that cutaneous phototoxic reactions to 5-MOP plus UVA are diminished by heat. Like 8-MOP, 5-MOP cross-links DNA and has the same skin photocarcinogenic potential as 8-MOP. Therefore the use of phototoxic psoralens in overthe- counter sunscreens is inappropriate because of the risk of increased UV-induced skin cancer.

Young A.R., Potten C.S., Chadwick C.A., Murphy G.M., Cohen A.J. (1988) "Inhibition of UV
radiation-induced DNA damage by a 5-methoxypsoralen tan in human skin" Pigment Cell Res.
1(5), 350-4 Abstract. Previously untanned buttock skin of 4 volunteers (skin type II; tan with
difficulty as they sunburn easily) was treated with various sunscreen preparations and solar--
simulated radiation (SSR) or SSR alone for 2 weeks. One week later, the treatment sites were
challenged with a DNA-damaging dose of SSR--twice the minimal erythema dose (2 MED). Skin
biopsy samples were assayed for the levels of unscheduled DNA synthesis (a measure of DNA
damage), melanin distribution, and skin thickening. 5-Methoxypsoralen-containing sunscreen
preparations plus SSR or SSR alone induced melanogenesis and increased the stratum corneum
thickness, but only the former regimen afforded a high degree of protection against subsequent
SSR-induced DNA damage. 5-Methoxypsoralen-free sunscreen preparations plus SSR induced
negligible tanning, skin thickening, and photoprotection. These findings are relevant to the riskbenefit analysis of sunscreen preparations, especially in skin type II, as they provide evidence that a 5-methoxypsoralen-induced tan is protective against the DNA-damaging effects of solar UV radiation, and thus has the potential to reduce the carcinogenic risk of exposure to such radiation.

Young A. R., Walker S.L., Kinley J.S., Plastow S.R. , Averbeck D., Morliere P., & Dubertret L.
(1990) “Phototumorigenesis studies of 5-methoxypsoralen in bergamot oil: Evaluation and
modification of risk of human use in an albino mouse skin model.” Journal of Photochemistry and
Photobiology B: Biology 7, 231-250. Abstract. The skin of the female hairless albino mouse (Skh
1) was used to study the enhancement of solar simulated radiation (SSR) tumorigenesis by 5-
methoxypsoralen (5-MOP) in model perfumes that contain bergamot oil. This work was done in
association with yeast mutagenicity studies and human skin phototoxicity studies. Analyses of
time-to-onset of tumour observation with 5-MOP at 0, 5, 15 and 50 ppm show a highly significant 5-MOP dose effect and the data indicate that 5-MOP has phototumorigenic potential even at 5 ppm. The addition of 0.5% UVB and 0.5% UVA sunscreens significantly reduces the
tumorigenicity associated with the vehicle (i.e. 5-MOP at 0 ppm) and 5-MOP at all concentrations.
Pairwise comparisons of 5-MOP (at 5 or 15 ppm) plus sunscreens with vehicle plus sunscreens
show that the sunscreens afford total protection at the lower 5-MOP concentrations. Additional
studies show that a 5-6 h delay between 5-MOP application and SSR exposure defers the timeto-onset of tumours as does intermittent 5-MOP and SSR treatment. A comparison of 5-MOP at 50 ppm in bergamot oil with 5-MOP at 50 ppm prepared from pure 5-MOP crystals shows
identical results, indicating that the active phototumorigenic agent in bergamot oil is 5-MOP and
not other related compounds, which may be present at greater concentrations. Analyses of
tumour histology at death show, in general, similar patterns of malignancy for all groups. Thus
although it is possible to delay tumorigenesis by various strategies, the tumours that eventually
develop are just as likely to be malignant, if not more so, when compared with non-delayed
groups.
 
Bioavailability of furanocoumarins.
Colombo, G., Zucchi A., Allegra F., Colombo P., Zani F. & Santi P. (2003) “In vitro and in vivo
study of 5-methoxypsoralen skin concentration after topical application.” Skin Pharmacology and
Applied Skin Physiology 16, 130-136.

Park Y. K., Hann S.K., Cho M.Y., Kim H.J., Shim S.C., Choi S.J. & M. H. Yun M.H.(1991) Study
of the skin concentrations after administration of the various phototoxic drugs. Yonsei Medical
Journal 32, 231-236.

Said A., Makki S., Muret P., Humbert P. & Millet J. (1997), Psoralens percutaneous permeation
across the human whole skin and the epidermis in respect to their polarity (in vitro study). Journal of Dermatological Science 14(2), 136-144.

Zucchi A., Raho E., Marconi B., Nicoli S., Santini M., Allegra F., Colombo P., Bettini R., & Santi P.
(2001) “Plasma and skin concentration of 5-methoxypsoralen in psoriatic patients after oral
administration.” Journal of Investigative Dermatology 117, 379-382.

(-) Byakangelicin.
CAS n°: 482-25-7
Occurs in roots of Ruta graveolens L. (Reisch et al. 1969). Byakangelicin angelate (Harmala et al. 1992); byakangelicin-2-O’-isovalerate (Sun & Jakupovic 1986) occur in roots of Angelica archangelica L.
Found at 97.89 ppm by UV-DAD in cold-pressed Californian lemon oil Citrus limonum (L.) (N. L. Burman) (Frerot & Decorzant 2004).
Byakangelicin is the corresponding diol formed from byakangelicol by hydration under acid conditions (e.g. in lemon oil by contact with lemon juice). Not particularly soluble in lemon oil; tends to crystallise out. Levels in essential oils therefore variable and processing-dependent.

Byakangelicol.
CAS n°: 26091-79-2
Found at 197.36 ppm by UV-DAD in cold expressed Californian lemon oil Citrus
limonum (L.) (N. L. Burman) (Frerot & Decorzant 2004).

Byakangelicol is an unstable epoxide compound which tends to hydrate to the
corresponding diol (byakangelicin) under acid conditions.

Frérot E. & Decorzant E. (2004) "Quantification of total furocoumarins in citrus oils by HPLC
coupled with UV, fluorescence and mass detection." J Agr. Food Chem. 52: 6879-6886.

Carrot seed oil. Daucus carota L. subsp. carota
CAS n°: 8015-88-1; EINECS-CAS n°: 84929-61-3
Cropwatch summary: Although a slight photosensitising effect is known from
whole carrots (Ceska 1986), carrot seed oil is not previously considered phototoxic.
Carrot seed oil not found photo-toxic by modified 3T3 neutral red uptake assay (Nathalie et al. 2006).

Ivie G.W., Beier R.C. & Holt D & L. (1982). "Analysis of the Garden Carrot (Daucus carota L.) for
Linear Furocoumarins (Psoralens) at the Sub Parts per Million Level." Journal of Agricultural and
Food Chemistry 30(3), 413-416. Abstract. Extracts of fresh roots and foliage of the garden carrot (Daucus carota L.) were analyzed by high performance liquid chromatography for the presence of linear furocoumarins (psoralens). Studies with samples fortified at sub-ppm levels with 12 psoralens of known structure, including psoralen, xanthotoxin, bergapten, isopimpinellin, heraclenin, and oxypeucedanin, showed that the procedure used was appropriate for the analysis of trace levels of psoralens in carrot. Results from studies with nonfortified samples showed that carrot does not contain these photosensitizing, photomutagenic, and photocarcinogenic chemicals or, if present, they occur at very low levels (0.5 ppm).

Nathalie D., Yannik G., Caroline B., Sandrine D., Claude F., Corrine C., Pierre-Jacques F. (2006)
“Assessment of the Phototoxic Hazard of Some Essential Oils using Modified 3T3 Neutral Red
Uptake Assay.” Toxicol in Vitro 20(4), 480-489.

Cedrat oil cold pressed Citrus medica L.
CAS n°: 68991-25-3.
Cropwatch summary: Peel oil claimed to contain negligible amount of FC’s
(Cropwatch private correspondence with certain Italian citrus oil manufacturers in
2007).

Celery seed oleoresin / oil. Apium graveolens L. var. dulce (Miller) DC.
CAS n°: 8015-90-5; EINECS CAS n° 89997-35-3
Cropwatch summary. FC’s known in stems, of plant, especially if diseased.
Seeds contain angelicin, sphondin, impertorium, bergapten, isopimpernellin, xanthotoxin, Dietry intake of celery (500g.) in an individual who visited a sun parlour afterwards resulted in severe burns (Ljunggren 1990).

Lombaert GA, Siemens KH, Pellaers P, Mankotia M, Ng W. (2001) "Furanocoumarins in celery
and parsnips: method and multiyear Canadian survey." J AOAC Int. 84(4):1135-43.
Abstract: The natural occurrence of biologically active furanocoumarins in common vegetables is an area of increasing interest with respect to human health. In this study, an efficient, rugged, and sensitive liquid chromatographic method with ultraviolet photodiode array detection was developed for the estimation of 5 biologically active furanocoumarins (psoralen, bergapten, xanthotoxin, trioxsalen, and angelicin) in celery and parsnips. When authentic samples were spiked with a mixture of furanocoumarins at individual levels of 2 to 10 microg/g, the method produced overall recoveries of 77 and 75% of all furanocoumarins from celery and parsnips, respectively. The method was applied in 2 laboratories to a multiyear survey of more than 200 samples. Of 110 parsnips samples, 109 (99%) contained quantitatable levels of furanocoumarins. The mean level of total furanocoumarins in the positive parsnip samples was 15.1 microg/g; the maximum level detected was 145 microg/g. Of 114 celery samples, 88 (77%) contained quantitatable levels of furanocoumarins. The mean level of total furanocoumarins in the positive celery samples was 1.9 microg/g; the maximum level detected was 15.2 microg/g. Xanthotoxin and bergapten were the most commonly detected furanocoumarins in both celery (68 and 63%) and parsnips (97 and 96%). Xanthotoxin had the highest mean level of positives in both celery (1.3 microg/g) and parsnips (8.5 microg/g). Little year-to-year variation in either total furanocoumarin levels or incidence was noted.

Citrus products - general.
The lagest contributors of FC’s to fragrances. For IFRA Standards for citrus oils,
see entry for bergapten above.

Bonaccorsi I.L., McNair H.M., Brunner L.A., Dugo P., Dugo G.(1999) "Fast HPLC for the analysis
of oxygen heterocyclic compounds of citrus essential oils." J Agric Food Chem. 47(10), 4237-9.
Abstract. A fast HPLC method for the determination of the oxygen heterocyclic compounds of
citrus essential oils was developed. Five different oils were analyzed under identical conditions,
by reversed-phase HPLC with photodiode array detector, for a direct comparison of the
composition of their oxygen heterocyclic fraction. Analysis time was 7 min. The oils analyzed
were lemon, bergamot, mandarin, sweet orange, and bitter orange. The method developed is
good for rapid screening or fingerprinting of these essential oils; a slightly slower method is
recommended for higher resolution and better quantitative results.

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.

Dugo, P.; Mondello, L.; Cavazza, A.; Dugo, G. (1995) “Oxygen heterocyclic compounds of citrus
essential oils.” Perfum. Flavor. 22, 25-30.
Dugo P., Mondello L., Sebastiani E., Ottanà R., Errante G.; Dugo G. (1999) "Identification of
minor oxygen heterocylic compounds of citrus essential oils by liquid hromatographyatmospheric pressure chemical ionization mass spectrometry." Journal of Liquid Chromatography & Related Technologies 22(19), 2991-3005
Abstract. The oxygen heterocyclic compounds (coumarins, psoralens, and polymethoxylated flavones (PMFs)) present in citrus essential oils (lemon, mandarin, sweet orange, bitter orange, bergamot, grapefruit, and lime) were analysed by an HPLC-MS system equipped with an APcI source. The mass spectra obtained at different voltages of the "sample cone" have been used to build a library. Citrus essential oils have been analysed with this system, using an optimised normal phase HPLC method, and the mass spectra were compared with those of the home made library. This method allowed the rapid identification and the characterisation of the oxygen heterocyclic compounds of citrus oils, the detection of some minor components for the first time in some oils, and the detection of authenticity and possible adulteration of the oils.
Dugo P, Mondello L, Dugo L, Stancanelli R, Dugo G.(2000) "LC-MS for the identification of
oxygen heterocyclic compounds in citrus essential oils." J Pharm Biomed Anal. 24(1), 147-54.
Abstract. The oxygen heterocyclic compounds (coumarins, psoralens and polymethoxylated
flavones) present in the nonvolatile residue of the essential oils of Mandarin, Sweet Orange, Bitter Orange, Bergamot and Grapefruit were analysed with an HPLC/API/MS system equipped with an APcI probe in positive mode. The use of hyphenated techniques, such as LC/MS provides a great information about the content and nature of constituents of natural complex matrices, such as essential oils. In this work, MS spectra were recorded at different voltages, to obtain structural information in addition to molecular weight information. The different response of the compounds identified has been also evaluated. The method allowed the confirmation of the identification of the main components of the fraction, previously reported for the different oils. MS characteristics of coumarins, psoralens and polymethoxylated flavones with different substitution patterns were determined on the basis of the response obtained with the APcI interface. Interface parameters were optimised to obtain a contemporaneous response for all the three classes of components.

Frérot E. & Decorzant E. (2004) "Quantification of total furocoumarins in citrus oils by HPLC
coupled with UV, fluorescence and mass detection." J Agr. Food Chem. 52: 6879-6886.
Abstract.
Furocoumarins or psoralens represent a class of photosensitizers whose use level is likely to be
restricted to 1 ppm in cosmetic products by the EU. A reversed-phase HPLC method was
developed to separate the 15 main furocoumarins present in citrus oils. Quantification by UV,
fluorescence, or mass detectors was compared in terms of linearity and limit of detection. Coldpressed oils of different citrus species were analyzed using this method. This method could be implemented in quality control laboratories equipped with an HPLC system and a UV diode array detector. Because of possible coelutions, the UV-spectral data should be carefully examined to avoid misleading interpretations of peaks.

Peroutka R., Schulzova V., Botek P. & Hajslova, J. (2007) "Analysis of furanocoumarins in
vegetables (Apiaceae) and citrus fruits (Rutaceae)." Journal of Food Scrence & Agriculture
87(11), 2152-2163.
Abstract. Several alternative approaches applicable for the analysis of furanocoumarins, toxic components occurring in some fruits and vegetables representing both Apiaceae and Rutaceae families, were tested in our study. Limits of detection (LODs) for angelicin, psoralen, bergapten, xanthotoxin, trioxsalen, isopimpinellin, sphondin, pimpinellin and isobergapten obtained by GC/MS (SIM) were in the range 0.01-0.08 g g-1. Slightly higher LODs (0.02-0.20 g g-1) were achieved by LC/MS-MS. The latter is the only alternative for analysis of bergamottin (LOD = 0.01 g g-1) in citrus fruits because this furanocoumarin is unstable under GC conditions. Regardless of the determination step used, the repeatability of the measurements (expressed as RSD) did not exceed 10%.

Saita T., Fujito H. & Mori M. (2004) "Screening of furanocoumarin derivatives in citrus fruits by
enzyme-linked immunosorbent assay." Biol Pharm Bull. 27(7), 974-7.
Abstract: This paper reports a sensitive and specific enzyme-linked immunosorbent assay for screening of furanocoumarin derivatives as cytochrome P450 3A4 inhibitors in citrus fruits. Anti-6',7'-dihydroxybergamottin antibody was obtained by immunizing rabbits with 6',7'-dihydroxybergamottin conjugated with bovine serum albumin using the N-succinimidyl ester method. An enzyme marker was similarly prepared by coupling 6',7'-dihydroxybergamottin with beta-D-galactosidase. The enzyme-linked immunosorbent assay is capable of detecting as little as 800 pg/ml of 6',7'-dihydroxybergamottin and 4 ng/ml of bergamottin. Cross-reactivity data showed that the antibody well recognizes both the furanocoumarin and 6,7-dihydroxy-3,7-dimethyloct-2-enyloxy moieties of the 6',7'- dihydroxybergamottin, and is thus specific to the structure of furanocoumarin derivatives containing geranyloxy side chain as the cytochrome P450 3A4 inhibitors in grapefruit juice. The antibody was, therefore, used for screening a large number of citrus fruits for furanocoumarin derivatives such as 6',7'-dihydroxybergamottin. Fifteen citrus fruits were examined and significant reactivity was observed in 8 of these: red pummelo, sweetie, melogold, banpeiyu pummelo, hassaku, sour orange, lime and natsudaidai. This enzyme-linked immunosorbent assay may be a powerful tool for screening for furanocoumarin derivatives as cytochrome P450 3A4 inhibitors in grapefruit juice.

Stanley W. & Jurd L. (1971) “Citrus coumarins.” J. Agr. Food Chem., 19, 1106–1110.
Abstract.
Coumarin compounds are generally distributed throughout the citrus species. The highest
amounts in number & concentration are found in lime oils, which contain about 7% by weight in
the cold-pressed oil; the lowest are found in orange oils which contain about 0.5%. The
physiological role of these compounds in the plants is not yet known, although there is reason to believe that they may be of importance. Closely related compounds, seselin & xanthyletin, were recently found in citrus roots and reported to inhibit certain enzyme systems. Substituted
furocoumarins have been shown to sensitise the skin to sunlight and to be toxic to fish. In
screening tests, derivatives of umbelliferone werrte found to exhibit antimicrobial activity primarily against yeast & moulds.

Clausena anisata leaf oil. Clausena anisata (Willd.) Hook f. ex Benth.
syn C. dentata (Willd.) M. Roemer; C. dunniana A. Leveille.
Cropwatch summary: Leaf oil contains FC’s such as xanthotoxin, bergapten (Okunade & Olaifa). This minor methyl-chavicol containing oil was previously promoted by the FAO, as a recommended minor cash crop for undeveloped countries. It is used to flavour the alcoholic liqueur Anisdos in the Philippines. However use of FC-containing plant extracts of Clausena anisata in Guatemala, Nigeria etc. to treat diseases may be related to the high incidence of skin cancer in these countries (Pieters & Vlietinek 1991).

Clementine terpenes (Italy) Citrus reticulata Blanco var. clementine.
EINECS CAS n° 68608-34-4
Cropwatch summary: No data.

(Perfume) Colognes.
Cropwatch summary: The term ‘berloque dermatitis’ (Zeynoun et al. 1981) arguably applies to this characterized adverse fragrance reaction especially when caused by 5-MOP from bergamot oil, whereas adverse dermatological reactions from plant constituents are often termed ‘phytophotodermatitis’.

Wang L, Sterling B, Don P.Cutis (2002). "Berloque dermatitis induced by "Florida water". Cutis
70(1), 29-30. Abstract. Phytophotodermatitis is a phototoxic dermatitis resulting from contact with psoralen-containing plants such as celery, limes, parsley, figs, and carrots. Berloque dermatitis is a variant of phytophotodermatitis and is caused by high concentrations of psoralen-containing fragrances, most commonly oil of bergamot. Berloque dermatitis is rarely seen today because of the removal of these fragrances from most cosmetic products in the United States. We report, however, a group of patients still at risk for berloque dermatitis. These patients use the colognes "Florida Water" and "Kananga Water," which are popular in Hispanic, African American, and Caribbean populations. These fragrant waters are used for spiritual blessing, treating headaches, and personal hygiene.

Zeynoun ST, Aftimos BA, Tenekjian KK, Kurban AK (1981) “Berloque dermatitis – a continuing
cosmetic problem.” Contact Dermatitis 7, 111-116.

Coriander leaf oil [syn Cilantro oil]. Coriandrum sativa L.
CAS n°: 8008-52-4; EINECS CAS n°: 84775-50-8
Cropwatch summary: Coriandrin (an isocoumarin with anti-HIV activity – see
Yu et al. 2003 below), bergapten & 8-MOP have been identified in leaves (45ug/g
leaves) & the fruit (Ceska et al 1987, 1988).

Coriander seed oleoresin / oil Coriandrum sativa L.
CAS n°: 8008-52-4; EINECS CAS n°:84775-50-8
Cropwatch summary: Fruits contain psoralen, angelicin etc. (Leung 1996). As coriander seeds are used in curries & cilantro leaves used in cooking, FC’s from these sources are ingested as a regular part if the diet (Kleiner et al. 2001).

Cumin oil Cuminum cyminum L.
CAS n°: 8014-13-9; EINECS CAS n°: 84775-51-9
Cropwatch summary: Cumin oil is restricted IFRA to 0.4% in final product for
leave-on products, on phototoxicity grounds. No restriction for wash-off products..

Dietry exposure to furanocoumarins.
Wagsaff (1991) estimated the US citizen’s average FC intake from diet at 1.3 mg/person/day with age-related uptake maximizing in the 16-20 age group at 2.48 mg/person/day. Whilst some authors ascribe an anti-carcinogenic effect to dietry FC’s, others question the role of dietry FC’s in increased melanoma incidence (Sayre & Dowdy 2008). Baumgart et al. (2005) showed furanocoumarins interfere with aryl hydrocarbon receptor-regulated CYP1A1
expression & activity in rat hepatocytes.

Baumgart, A. Schmidt, M. Schmitz, H. J. Schrenk, D. (2005) "Natural furocoumarins as inducers
and inhibitors of cytochrome P450 1A1 in rat hepatocytes." Biochemical Pharmacology 69(4),
657-667 Abstract: Furocoumarins are natural plant constituents present in medicinal plants and in a variety of foods such as grapefruit juice. They are phototoxic and act as potent inhibitors of drug metabolism. We have investigated the interaction of four furocoumarins angelicin, bergamottin, isopimpinellin, and 8-methoxypsoralen with the expression and activity of aryl hydrocarbon receptor (AhR)-regulated CYP1A1 in rat hepatocytes in primary culture, both in the presence and absence of light. In intact hepatocytes pretreated with 2,3,7,8- etrachlorodibenzo-p-dioxin and in microsomes isolated thereof, all furocoumarins tested acted as potent inhibitors of CYP1A1 activity bergamottin being the most potent inhibitor in microsomes with an IC50 of 10nM in the presence and 60nM in the absence of light. 8-Methoxypsoralen and angelicin led to a significant induction of CYP1A1 mRNA in hepatocytes, while all furocoumarins except bergamottin increased xenobiotic-responsive element-driven reporter gene expression in transfected H4IIE rat hepatoma cells when light was excluded. Furthermore, all furocoumarins tested induced the expression of endogenous, immunoreactive CYP1A1 protein, primarily in the dark. In conclusion. our results demonstrate that individual furocoumarins present in food and medicinal plants can interfere with AhR-regulated CYP1A1 expression and activity in at least three major ways, i.e., (i) act as highly potent inhibitors of the catalytic activity of CYP1A1 both in the presence and absence of light, (ii) induce CYP1A1 gene expression in the absence of light via activation of the AhR, and (iii) induce CYP1A1 gene expression without activation of the AhR.

Sayre R.M. & Dowdy J.C. (2008) "The increase in melanoma: Are dietary furocoumarins
responsible?" Med Hypotheses. 70(4), 855-9.
Abstract. According to most cancer registries the incidence of cutaneous melanoma (CM) has been increasing for several decades. Unlike other skin cancers, CM does not clearly correlate with exposure to ultraviolet radiation. The strongest etiological evidence for CM in man is genetic predisposition, evidenced by very high risks in primary relatives of melanoma patients, and photochemotherapy with 8-methoxy psoralen in combination with ultraviolet-A radiation (PUVA) to treat psoriasis and vitiligo. Retrospective studies of PUVA patients show significantly increased incidence of CM. Psoralens, and other furocoumarins, are phototoxic and photocarcinogenic, intercalate DNA and photochemically induce mutations. Furocoumarins are botanical phytoalexins found to varying extents in a variety of vegetables and fruits, notably citrus fruits. The levels of furocoumarins present in our diets, while normally well below that causing evident acute phototoxicity, do cause pharmacologically relevant drug interactions. For the past approximately 50 years CM has increased at similar rates as the increased availability and consumption of citrus products. Recently in a large study of nurses, only orange juice drinking, indicative of dietary preference for citrus, was positively associated with significantly increased risk of developing CM. We hypothesize that the increases in cutaneous melanoma incidence may be in part related to concomitant increases in dietary photocarcinogenic furocoumarins.

Schlatter, J., B. Zimmerli, R. Dick, R. Panizzon, and C. Schlatter (1991) “Dietary intake and risk
assessment of phototoxic furocoumarins in humans.” Food and Chemical Toxicology 29(8), 523-
530. Abstract. The question of whether the furocoumarin content of vegetables is sufficient to
induce phototoxic skin reactions after ultraviolet irradiation was examined in two experiments with our human volunteers. In a first experiment, 300 g of celery roots (total phototoxic furocoumarin content 28.2 micrograms/g) was ingested. No skin reactions were observed after UVA exposure (1.5-9 J/cm2), and the blood levels of the furocoumarins--psoralen, 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen (5-MOP)--were below the analytical detection limit of 2 ng/ml. To investigate the phototoxic effects of a mixture of the two most important furocoumarins in vegetables, after gastro-intestinal uptake, 8-MOP and 5-MOP (15 mg each) were ingested separately in a 50% alcoholic solution. A strong and persistent erythema was induced in three out of the four subjects (UVA dose: 3-25 J/cm2). The blood levels immediately before UVA irradiation varied between 14 and 114 ng/ml (8-MOP), and 17 and 70 ng/ml (5-MOP). In the subject who did not show phototoxicity, the blood levels remained at trace levels (3 ng/ml). Two subjects were also tested with a mixture of 10 mg 8-MOP plus 10 mg 5-MOP; in one subject the mixture induced pigmentation only, while in the other a mild-to-medium erythema was induced. The blood levels of the furocoumarins in the two subjects were similar (12-15 ng/ml for 8-MOP and 5-MOP).
It is concluded that in humans the phototoxic threshold dose of furocoumarin mixtures is of the
order of 10 mg 8-MOP plus 10 mg 5-MOP, which is equivalent to about 15 mg 8-MOP per person
(blood levels of 8-MOP and 5-MOP at 30 min after ingestion of about 10-15 ng/ml each). This
phototoxic threshold dose was not reached by the consumption of celery roots and other
conventional vegetables under normal dietary habits (experimental intake of 2-8 mg per subject of the potentially phototoxic furocoumarin mixture). However, the safety factor between the possible actual intake of furocoumarins and the phototoxic threshold dose is about 2-10, which is relatively small.

Dill seed / herb oils. Anethum graveolens L.
CAS n°: 8005-75-5; EINECS CAS n°: 90028-03-8
Cropwatch summary: Bergapten has been identified in dill fruits (Dranik & Propokenko 1969). Plant parts also contain xanthotoxin, but the FC content believed to be low.

Stavri M, Gibbons S. (2005) "The antimycobacterial constituents of dill (Anethum graveolens)."
Phytother Res. 19(11):938-41. Abstract. As part of a project to characterize selected members of the Kuwaiti flora for their phytochemistry and antimycobacterial activity, a new furanocoumarin, 5-[4''-hydroxy-3''-methyl-2''-butenyloxy]-6,7-furocoumarin (3), was isolated from the whole herb of Anethum graveolens. The known compounds oxypeucedanin (1), oxypeucedanin hydrate (2) and falcarindiol (4) were also isolated from this plant. The structure of each compound was determined by interpretation of NMR and mass spectrometric data. The three known compounds exhibited antibacterial activity against a panel of rapidly growing mycobacteria with minimum inhibitory concentration (MIC) values in the range 2-128 microg/mL.

Dittany herb oil (syn. Gas Plant) Dictamnus albus L.
CAS n°: 89998-27-6 EC n°: 289-766-7
Cropwatch summary: FC’s known to occur in plant. Epoxybergamottin
Epoxybergamottin is present in grapefruit peel & is a CYP3A4 inhibitor (the oral availability of many drugs metabolised by the enzyme cytochrome P(450) 3A4 (CYP3A4) is increased if co-administered with grapefruit juice): Wangensteen et al (2003). In grapefruit juice processing, the natural epoxybergamottin may be largely hydrolysed to 6,7-dihydroxybergamottin, another important CYP3A4 inhibitor.
Epoxybergamottin has been found at 1732.1 ppm in cold-pressed Israeli grapefruit oil Citrus paradisi Macf., & and at 814.2 ppm in cold-pressed bitter orange oil (Frerot & Decorzant 2004).
Epoxybergamottin is known for its ability to inhibit aromatase, which is associated
with the progression of breast and prostate cancer.

Erythema.
Auletta M., Gange R.W., Tan O.T. & Matzinger E. (1986) "Effect of Cutaneous Hypoxia upon
Erythema and Pigment Responses to UVA, UVB, and PUVA (8-MOP + UVA) in Human Skin."
Journal of Investigative Dermatology 86, 649–652.
Abstract. The effect of oxygen deprivation upon UVA-, UVB-, and PUVA- induced pigment and erythema responses in normal human skin was examined. Before exposure, varying degrees of hypoxia in the skin of the forearm were achieved by inflating sphygmomanometer cuff applied to the upper arm. After the transcutaneously measured pO2 had stabilized, sites on the inner forearm were exposed to UVA, UVB, or 8-MOP + UVA radiation, to determine dose thresholds for the induction of erythema and pigmentation at different cuff pressures. Inflation of the cuff to greater than systolic pressure completely inhibited immediate and delayed pigment responses (IPD, DT) to UVA doses greater than 10 times the normal pigmentation threshold dose. UVA-induced delayed erythema responses were partially inhibited by cuff inflation: 2.7 times the minimal erythema dose of UVA was necessary to cause an erythema response when exposure occurred during vascular occlusion. In contrast, erythema and pigments responses to UVB and PUVA were unaltered by cuff pressures exceeding systolic pressure during exposure. Inhibition of UVA-induced erythema and pigment responses by vascular occlusion were reversed by the transcutaneous diffusion of 100% O2. These findings indicate that the cutaneous responses to UVA and UVB occur by separate pathways differing with respect to O2-dependence. Our findings agree with those of other studies which indicate that PUVA-induced phototoxicity and melanogenesis are not O2- dependent.

Barolet D, Boucher A. (2008) "LED photoprevention: Reduced MED response following multiple
LED exposures." Lasers Surg Med. 40(2), 106-12.
Abstract. BACKGROUND AND OBJECTIVES:
As photoprotection with traditional sunscreen presents some limitations, the use of non-traditional reatments to increase skin resistance to ultraviolet (UV) induced damage would prove particularly appealing. The purpose of this pilot study was to test the potential of non-thermal pulsed light-emitting diode (LED) treatments (660 nm) prior to UV exposure in the induction of a state of cellular resistance against UV-induced erythema.
STUDY DESIGN/MATERIALS AND METHODS: Thirteen healthy subjects and two patients with polymorphous light eruption (PLE) were exposed to 5, 6, or 10 LED treatments (660 nm) on an EXPERIMENTAL anterior thigh region. Individual baseline minimal erythema doses (MED) were then determined. UV radiation was thereafter performed on the LED EXPERIMENTAL and CONTROL anterior thigh areas.
Finally, 24 hours post-UV irradiation, LED pre-treated MED responses were compared to the nontreated sites. RESULTS: Reduction of erythema was considered significant when erythema was reduced by >50% on the LED-treated side as opposed to CONTROL side. A significant LED
treatment reduction in UV-B induced erythema reaction was observed in at least one occasion in
85% of subjects, including patients suffering from PLE. Moreover, there was evidence of a doserelated pattern in results. Finally, a sun protection factor SPF-15-like effect and a reduction in post-inflammatory hyperpigmentation were observed on the LED pre-treated side.
CONCLUSIONS: Results suggest that LED based therapy prior to UV exposure provided
significant protection against UV-B induced erythema. The induction of cellular resistance to UV
insults may possibly be explained by the induction of a state a natural resistance to the skin via
specific cell signaling pathways and without the drawbacks and limitations of traditional
sunscreens. These results represent an encouraging step towards expanding the potential
applications of LED therapy and could be useful in the treatment of patients with anomalous
reactions to sunlight.

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.

Fig leaf absolute. Ficus caria L.
CAS n°: 68916-52-9; EINECS-CAS n°: 90028-74-3
Cropwatch summary. Fig leaf absolute is banned IFRA on phototoxicity & sensitization grounds. Exposure to the milky latex from fig leaves followed by sun exposure causes phytodematitis to 10% of fig handlers in Turkey. The sap contains up to 2090μg/ml psoralens including psoralen & bergapten (Beier & Nigg 2001).

Grapefruit oil, white, cold-pressed - all origins. Citrus paradisi Macf.
CAS n°: 8016-2-4; EINECS-CAS n° 90045-43-5.
Cropwatch summary:
Phototoxicity. Although Sams (1941) & Opdyke (1974) previously claimed it was non-photo-toxic, it is now restricted IFRA to 4% concentration in fragranced products not washed off skin exposed to sunshine.
Composition. Cold pressed white grapefruit oils contain 1.4% to 1.5% FC’s (mainly) including bergamottin & epoxybergamottin. Some varieties of ‘Sweetie’ grapefruit oils also contain FC’s.
RIFM give the following data “measured by the fragrance industry” (??) for grapefruit oil (but no botanical or geographical source details given): bergapten up to 1000ppm, isopimpernellin <5ppm, bergamottin up to 1100 ppm, epoxybergamottin 1120 ppm; psoralen, xanthotoxin, oxypeucedanin & angelicin not detected (RIFM Fact Sheet No 3.).
This data is in apparent conflict with that of Frerot & Decorzant (2004) who found 700.2 ppm oxypeucedanin in cold-pressed Israeli Grapefruit oil by UV-DAD.
Previously McHale & Sheridan (1989) had found 2000 ppm of bergamottin & 9500 ppm epoxybergamottin in cold-pressed grapefruit oil, whereas Dugo et al. 1997 had found 970 ppm of bergamottin & 11,260 ppm epoxybergamottin in cold-pressed grapefruit oil.
Furanocoumarins such as bergamottin & 6’,7’-dihydroxybergamottin & furanocoumarins dimers (& flavonoids such as naringen & naringenin) from grapefruit juice have been found responcible in certain instances for the disruption of the oral availability of prescription drugs in patients via the inhibition of the cytochrome P-450 (CYP3A4) system in the small intestine.

Hyperpigmentation
d'Ischia M, Napolitano A, Prota G. (1989) “Psoralens sensitize glutathione photooxidation in
vitro.” Biochim Biophys Acta. 993(2-3), 143-7. Abstract. In vitro experiments are reported showing that psoralens and other furocoumarins of current pharmacological interest, e.g., angelicin and 4,6,4'-trimethylangelicin, all have, to a variable extent, the ability to sensitize the photooxidation of glutathione in ethanol/phosphate buffer with pyrex-filtered ultraviolet light. Besides substrate concentration and the nature of the furocoumarin used, the rate of the sensitized reaction is markedly dependent on the partial pressure of oxygen and the pH of the medium, being progressively faster on passing from pH 5 to pH 8.5. Scavengers of superoxide ions (superoxide dismutase), hydrogen peroxide (catalase) and singlet oxygen (sodium azide, diazabicyclooctane, sorbic acid) have little or no inhibitory effect on the reaction rate. These and other data suggest that furocoumarins can directly sensitize glutathione photooxidation by forming a charge transfer complex which is driven to the oxidized products in the presence of oxygen. The possible relevance of these results to the mechanisms of skin melanin hyperpigmentation induced by furocoumarins and ultraviolet light is discussed.

Imperatorin.
CAS n°: 482-44-0
16.24 ppm imperatorin found by UV-DAD in cold pressed Californian lemon oil Citrus limonum (L.) (N. L. Burman) (Frerot & Decorzant 2004). Imperatorin is also found in lime oil, parsnip & parsley plant parts etc. The alleged absence of imperatorin (together with the absence of 7-geranoxycoumarin) from lemon oil has been suggested as a mehod of detecting “cross-contamination” (Cropwatch calls it adulteration!).

Imperatorin shows anti-carcinogenic effect against chemically induced cancer in a murine model (Prince et al. 2006). Imperatorin also inhibits drug metabolizing enzymes (Woo et al. 1993) & blocks mutagenesis by B[a]P in Salmonella typhimurium in the presence of a hepatic 9000 g supernatant (S9) activating system (Wall et al. 1990). Imperatorin & isopimpinellin may thus have anticarcinogenic effects when administered in diet (Kleiner et al. 2001); the authors
have suggested this is due to a balance between P450 inhibitory activity and an ability to induce glutathione-S-transferase (GST) production.

Kleiner H.E., Vulimiri S.V., Miller L, Johnson W.H. Jr., Whitman C.P. & DiGiovanni J. (2001) "Oral
administration of naturally occurring coumarins leads to altered phase I and II enzyme activities
and reduced DNA adduct formation by polycyclic aromatic hydrocarbons in various tissues of
SENCAR mice." Carcinogenesis 22(1), 73-82.
Abstract. Several naturally occurring coumarins, to which humans are routinely exposed in the diet, were previously found to inhibit P450-mediated metabolism of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) in vitro, block DNA adduct formation in mouse epidermis and inhibit skin tumor initiation by B[a]P and/or DMBA when applied topically to mice. The present study was designed to investigate the effects of two of these compounds, of the linear furanocoumarin type, when given orally (70 mg/kg per os, four successive daily doses), on P450 and glutathione S-transferase (GST) activities and DNA adduct formation by B[a]P and DMBA in various mouse tissues. Imperatorin and isopimpinellin significantly blocked ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O:-dealkylase (PROD) activities in epidermis at 1 and 24 h after oral dosing. Imperatorin and isopimpinellin modestly inhibited EROD activities in lung and forestomach at 1 h and significantly inhibited PROD activities in lung and forestomach at 1 h after the final oral dose. Twenty-four hours after the final oral dose of imperatorin or isopimpinellin EROD and PROD activities remained inhibited in epidermis and lung. However, forestomach P450 activity had returned to control levels. Interestingly, imperatorin and isopimpinellin treatment inhibited liver EROD activity at 1 h, had no effect on PROD activity at this time point, but elevated both these enzyme activities at 24 h. Elevated EROD and PROD activities coincided with elevated hepatic P450 content.
Imperatorin and isopimpinellin treatment also increased liver cytosolic GST activity at both 1 and
24 h after the final oral dose by 1.6-fold compared with corn oil controls. Oral administration of
imperatorin and isopimpinellin also had a protective effect against DNA adduct formation by B[a]P and DMBA. Imperatorin pretreatment decreased formation of DNA adducts by DMBA in
forestomach. Pretreatment with isopimpinellin led to reduced DNA adduct levels in liver (B[a]P),
lung (B[a]P) and mammary epithelial cells (DMBA). These results suggest that imperatorin and
isopimpinellin may have potential chemopreventive effects when administered in the diet.

Prince M., Campbell C.T., Robertson T.A., Wells A.J. & Kleiner H.E.(2006) "Naturally occurring
coumarins inhibit 7,12-dimethylbenz[a]anthracene DNA adduct formation in mouse mammary
gland." Carcinogenesis. 27(6), 1204-13. Abstract. Naturally occurring coumarins (NOCs) are anticarcinogenic in the mouse skin model. To characterize the chemopreventive potential of NOCs against breast cancer, we first examined their effects on 7,12-dimethylbenz[a]anthracene
(DMBA)-DNA adduct formation in mouse mammary gland. We hypothesized that those NOCs
that both inhibited cytochrome P450 1A1/1B1 and induced hepatic glutathione S-transferases
(GSTs) would be the most effective in blocking DMBA-DNA adduct formation in mouse mammary
gland. To address this hypothesis, simple coumarins (e.g. coumarin and limettin, which induced
mouse hepatic GSTs but had little effect on P4501A1/1B1) and linear furanocoumarins (e.g.
imperatorin and isopimpinellin, which induced hepatic GSTs and were potent inhibitors of
P4501A1/1B1) were compared. Mice were pretreated with NOCs (150 mg/kg body wt, by gavage) prior to either a single dose of DMBA (50 microg) or multiple doses of DMBA (20 microg daily for 3 and 6 weeks). Mammary DMBA-DNA adduct formation was quantitated by the nuclease P1-enhanced 32P-postlabeling assay. With the single dose of DMBA, coumarin, limettin, imperatorin and isopimpinellin inhibited DMBA-DNA adduct formation by 50, 41, 79 and 88%, respectively.
Coumarin, limettin and imperatorin blocked DMBA-DNA adduct formation by 36, 60, and 66% at 3
weeks, and by 0, 49 and 55% at 6 weeks of DMBA dosing, respectively. In a 6 week doseresponse study of select NOCs and 7,8-benzoflavone (a potent P4501 inhibitor that had little effect on GSTs), DMBA-DNA adduct formation was inhibited by 0, 43 and 24% in the limettin
groups; by 26, 26 and 69% in the isopimpinellin groups; and by 80, 96 and 97% in the 7,8-
benzoflavone groups at 35, 70 and 150 mg/kg, respectively. Taken together, these results
suggest that linear furanocoumarins had a greater inhibitory effect on DMBA-DNA adduct
formation in mouse mammary glands compared with simple coumarins, and that the predominant effect may be P4501 inhibition.

Isoimperatorin
CAS n°: 482-45-1
42.86 ppm isoimperatorin found by UV-DAD in cold pressed Californian lemon oil Citrus limonum (L.) (N. L. Burman) (Frerot & Decorzant 2004). Isoimperatorin (& imperatorin) separated from the Chines drugs Tang-Bai-Zai & Ashita-ba show strong anti-tumour activity in cultured cells (Okuyama et al. 1990).

Isopimpinellin.
CAS n°: 482-27-9
Linear furanocoumarins, present in rind & pulp of limes (S furanocoumarins 3μg/g pulp; 5 mg/g in oil: Nigg et al 1993) and to 1.3 ppm in celery (Beier et al. 1983).
Isopimpinellin as present in lime oil was previously thought to be non-photoactive (Musajo et al. 1953), and any photo-toxicity associated with isopimpinellin has been more recently disputed (Ivie & Beier 1996) – any previous alleged activity (Ivie 1978) was probably due to impurities in materials used.
Isopimpinellin shows anti-cancer effects with chemically induced tumours (Kleiner et al. 2002). Altamirano-Dimas et al. (1986) suggest that isopimpinellin induces cross-linking in viral DNA. However there was no demonstrable phototoxicity towards bacteriophage T4, the murine cyclomegalovirus & the Sindbis virus.

The SCCNFP presented an Opinion on isopimpinellin (SCCNFP/0761/03) disclosing a concentration of 2000 ppm in cold pressed lime oil (type, botanical & geographic origin not stated) and 200 ppm in rue oil (geographic botanical origin not stated). The SCCP concluded that submitted data demonstrated that ‘isopimpinellin is photomutagenic in Salmonella typhimurium TA102’. Cropwatch comments: how can we tell? Although some details were reproduced in the Opinion, a complete published paper does not appear to available in the public domain. The SCCP also concluded that there was incomplete information on
photo-mutagenicity and on photo-clastogenicity of isopimpinellin to enable a safety evaluation.

Kleiner H.E., Reed M.J. & DiGiovanni J. (2003) “Naturally occurring coumarins inhibit human
cytochromes P450 and block benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene DNA adduct
formation in MCF-7 cells.” Chem Res Toxicol. 16(3), 415-22. Abstract. Naturally occurring
coumarins (NOCs) inhibit polycyclic aromatic hydrocarbon-induced skin tumor initiation in mice by
blocking cytochrome P450 (P450)-mediated bioactivation of benzo[a]pyrene (B[a]P) and 7,12-
dimethylbenz[a]anthracene (DMBA). Bergamottin selectively inhibits tumor initiation by B[a]P,
whereas imperatorin and isopimpinellin inhibit tumor initiation with both carcinogens. The goals of the current study were to examine the ability of NOCs to inhibit human P450s in vitro and to
establish whether NOCs, which are anticarcinogenic in mice, can block carcinogen bioactivation
in cultured human cells. For the initial experiments, incubations containing 5 microM P450, P450
substrate, an NADPH generating system, and NOCs were used to determine the concentrations
of each inhibitor that blocked 50% of P450 activity (IC(50)). These results confirmed that NOCs
are capable of inhibiting multiple human P450s and that they exhibit selectivity for certain
isoforms of human P450s. In subsequent experiments, we examined the effects of bergamottin,
imperatorin, and isopimpinellin on DMBA and B[a]P DNA adduct formation in the human breast
MCF-7 adenocarcinoma cell line. Coincubation of cells with the three different NOCs significantly
inhibited DMBA DNA adduct formation by 29-82% at doses ranging from 2 to 10 microM and
significantly inhibited B[a]P DNA adduct formation by 37-80% at doses ranging from 20 to 80
microM. HPLC analysis of the DNA hydrolysates demonstrated that inhibition of DNA adducts
corresponded to inhibition of the major B[a]P and DMBA diol-epoxide-derived adducts. Although
bergamottin was not effective at blocking DMBA bioactivation in the mouse skin model, it was
similar in effectiveness to imperatorin and isopimpinellin in MCF-7 cells. These results
demonstrate that NOCs, which are present in citrus fruits and other components of the human
diet, are capable of inhibiting carcinogen metabolizing enzymes and blocking bioactivation of both B[a]P and DMBA in MCF-7 cells.

Knudson E.A. & Kron S (1988) “In vitro and in vivo phototoxicity of furocoumarin-containing
plants” Clinical and Experimental Dermatology 13 (2), 92–96. Abstract. The purpose of the study was to investigate the individual phototoxic reactivity in the skin to a series of plants, selected for their phototoxicity as determined in the Daniel's in vitro test. Twenty volunteers of skin type II, nine females and eleven males with normal sunlight reactivity were included in the study. Juice and leaves of wild parsnip, giant hogweed, angelica, garden parsley, celeriac and dill were applied on unpigmented skin of the thighs and irradiated after 1–11/2 h with 2 and 4 J/cm2 UVA. The study showed clear ranking of the tested plants with wild parsnip and giant hogweed as the most and celeriac and dill as the least potent Doubling of the UVA dose resulted in more and stronger erythema reactions. Hydration (occlusion) of the test substances had a similar promotion effect. Tests with leaves gave stronger reactions than those with juices. Isolated pigment reactions were often seen when the combined doses of furocoumarins and UVA were too weak to cause erythema. These reactions could be compared with Berloque dermatitis. Such pigment reactions are easily overlooked and could be one of the explanations for the few observed cases of phytophotodermatitis in the dermatological clinic.

Musajo L., Rodighiero G. & Caporale G. (1953) Chim. Ind. 35, 13.

Nigg H.N., Nordby H.E., Beier R.C., Dillman A., Macias C., & Hansen R.C. (1993) “Phototoxic
coumarins in limes” Food Chem Toxicol 31, 331-335. Abstract: Coumarins in the rind and pulp of
Persian and Key limes were quantified. In the rind of Persian limes, coumarin concentrations
were in the order: limettin > bergapten > isopimpinellin > xanthotoxin > psoralen. In the rind of
Key limes, psoralen and xanthotoxin were analytically absent; limettin was 10 times more
concentrated than either bergapten or isopimpinellin, which were equal in concentration.
Coumarin content in Persian lime pulp was in the order; isopimpinellin > limettin > bergapten >
xanthotoxin > psoralen.

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