Gielen Aroma

Furanocoumarins T. Burfield

www.cropwatch.org
THE FIRST TRULY INDEPENDENT WATCHDOG FOR THOSE
WORKING WITH NATURAL AROMATIC MATERIALS
E: info@cropwatch.org T: ++44 (0)7771 872 521
Furanocoumarins – Properties & their Distribution in
Natural Aromatic Ingredients: an A-Z Listing.
Copyright Ó Cropwatch Mar 2008 v 1.00
[N.B. Some references are duplicated under a number of subject headings].

Pre-amble.
1. Furanocoumarins (syn. furocoumarins: FC’s) are found distributed within species from approximately five main plant families according to Daniel et al. (1999), these being the Apiaceae (Umbelliferae), Moracea, Rutaceae, Fabaceae, & the Leguminaceae, although according to (Aucoin et al. 1992) FC’s have additionally been found in the Solanaceae, Asteraceae, Pittosporaceae & the Thymelaceae. In combination with long-wavelength UVA, they may trigger adverse phototoxic effects, but not all FC’s are phototoxic, and not all phototoxic
properties of essential oils are down to any contained FC’s. Moreover, no single in vitro test currently exists which can predict the photo-carcinogenicity of furanocoumarins. FC’s can be divided into linear furanocoumarins (psoralens) or angular furanocoumarins (angelicins); angular furanocoumarins normally only produce DNA-adducts, whereas linear furanocoumarins produce DNA-adducts and the potentially more toxic DNA-DNA interstrand cross links. Citrus oils
represent the largest FC contributors to fragrances (Frérot & Decorzant 2004), and the importance of maintaining the availability of citrus oil ingredients within the fragrance art has been previously described (Cropwatch 2007 – see http://www.cropwatch.org/nlet6.htm).

2. Psoriasis affects approximately 2% of the population (Disepio et al. 1999).
Administration of FC’ s orally or topically, the latter effected by bathing in a diluted FC solution or by applying psoralen-containing creams, followed by UVA radiation in the range 320-400 nm range, is termed ‘PUVA’. It has been described thus: “(PUVA) is now a widely used, frequently convenient and effective systemic treatment of psoriasis with well-characterised and controllable side effects” [Lewis (1994); McNeely & Goa (1998); through Ojala (2001)]. Most
information on the photo-carcinogenic effects on the common FC’s: 5-MOP & 8-MOP come from PUVA studies (DoH 1998), There have been many studies investigating the effects of repeated PUVA treatments over time, carried out, as they are, with single (& sometimes impure) furanocoumarins sometimes in conjunction with crude coal tar, under exaggerated UV exposure conditions on a subsection of the population with compromised skin. It does appear that some
studies indicate that long-term PUVA treatment with 8-MOP can cause persistent pigmentation & other skin changes. Further, it is now more recently muted that PUVA treatment causes squamous cell skin cancer in a dose- and timedependent fashion, relative risks being about roughly from 6- to 12-fold after about 8-10 years follow-up (Hannuksela-Svahn 2000; Stern 2007). However new improvements in PUVA therapy technique are reported which may help reduce adverse effects (Grundmann-Kollman et al. 2001, Man et al. 2004). It also has to
be remembered that the actual mechanism of PUVA therapy is not understood (Viola et al. 2008), although some investigative attempts to elicidate the sequence of photobiophysiological sequence of events have been made (Potapenko AIa et al. 2004). Pathak et al. (1983) found that the photoconjugation of DNA with photoreactive ingredients of crude coal tar caused interstrand crosslinks, and also that crude coal tar is a more efficient producer of the reactive
oxygen species 1O2 than 8-methoxypsoralen, and is responsible for cellmembrane damage and cellular edema. However, Stern (1994) subsequently ruled out topical tar as factor in squamous cell carcinoma (SCC), but ruled in PUVA & methotrexate as causing increases in patent incidence of SCC.

3. Furanocoumarins absorb UV radiation, putting electrons into higher energy levels. When returning to ground levels, energy is retransmitted as light and the transference of energy from electrons to other molecules results in the phototoxic effect. According to the exact conditions (concentration of FC, magnitude of dose of UVA etc.), the phototoxic effect can manifest as covalent binding of FC’s to DNA or cell protein constituents, or the production of damaging energetic oxygen spp., or the binding of photogenerated species to various receptors etc.
Physically this can result in skin pigmentation, erythema, edema, photo-irritation,
photo-mutagenic events etc.

4. Risk-benefit concerns. Furanocoumarins, especially 5-MOP & 8-MOP, have historically been used for photochemoprotection, especially with UVB sunscreens (Fitzpatrick 1989, Kligman & Forlot 1989), and several FC-containing essential oils, or FC’s themselves show anti-carcinogenic effects (Prince et al. 2006; Bakkali et al. 2008). Some essential oils have useful anti-oxidant properties which can act against damaging reactive oxygen species generated by skinexposed UV-light (Sakurai et al. 2005). According to some commentators, dietry
FC intake itself is believed to impart anti-carcinogenic benefits (Kleiner et al. 2001), and many FC’s are capable of inhibiting carcinogen-metabolising enzymes (Cai et al. 1997a; Cai et al. 1997b; Kleiner et al. 2003), or blocking tumor formation, but others question the role of dietry FC’s in increased melanoma incidence (Sayre & Dowdy 2008). Schlatter et al. (1991) point out that “the safety factor between the possible actual (dietry) intake of furocoumarins and the phototoxic threshold dose is about 2-10, which is relatively small”, but for some reason, EU regulation of furanocoumarins exposure from foodstuffs (e.g. from cooked parsnips, celery, parsley, spices, citrus fruits & juices, lime marmalade etc. etc.) does not occur in a similar manner to that proposed for cosmetics.
The overall risk-benefit effects for many individual FC-containing natural complex substances is therefore complex, and at present, still imperfectly understood. Proposed EU legislation to limit FC’s in cosmetics would therefore be overhasty & inappropriate, although labelling advice (say, to apply FC-containing cosmetics not to be washed off the skin only under heavy clothing, or where skin is not to be exposed to actinic light for 12-24h.), would be worth considering.

5. Ford (1991) listed the following aroma materials that cause phototoxic contact dermatitis:
[5-acetyl-1,1,2,3,3,6-hexamethylindane] Angelica root oil (Angelica archangelica L.)
Bergamot oil expressed (Citrus aurantium L. bergamia Wright et Arn.) Fig Leaf absolute (Ficus caria) Lemon oil cold pressed (Citrus limon L. Burn. F.) Lime oil expressed (Citrus aurantifolia Swingle) [N.B. Citrus latifolia not mentioned! TB] Orange peel oil, bitter (Citrus aurantium L.)
Rue oil (Ruta graveloens L.) Tagetes absolute (Tagetes patula L.) Tagetes minuata absolute (Tagetes minuata or T. glandulifera) Tagetes oil (Tagetes erecta L., T. glandulifera Schrank)
Verbena oil (Lippia citriodora Kunth).

6. Phototoxicity testing has historically used animal models, raising ethical issues. More recent in vitro testing protocols feature test models including primary lymphocytes & cultured mammalian cell lines. The 3T3 mouse fibroblast model (Duffy et al. 1987) was developed & validated in a joint project of the EU & COLIPA (Spielmann et al. 1994, 1996), and the technique has thrown up positive results for phototoxicity for essential oils previously considered non-phototoxic (Dijoux et al. 2006). It is worth repeating however that no single in vitro test
currently exists which can predict the photo-carcinogenicity of chemical substances, furanocoumarins or otherwise. The current confusion over the alleged photo-toxicity of some Tagetes qualities (see accompanying document) where career toxicologists (from REXPAN) have pre-decided the answer they want, but can’t find the methodology which will give it to them, easily describes the farce that the essential-oil producing/using community is caught up in.

References.
In verband met de uitgebreidheid zal ik wat references verwijderen. Indien daar behoefte aan bestaat, kan het originele document worden gemaild.

Furanocumarins in natural aroma ingredients: an A-Z listing.

Ammi majus extract Ammi majus L.
Cropwatch summary: Described in older Arabic literature as local medicinal reatment for leukodermia – fruits consumed orally and leukodermic patches exposed to sunshine (Dawood El-Antaki 1923). First used as source of xanthotoxin for vitiligo treatment (Mofty 1948). Ingredient hardly used in Western corporate perfumery.

Dawood El-Antaki (1923) Tazkaret Oli El-Albab 3rd editton, Vol 1, 32.
Hehmann M., Lukacin R., Ekiert H. & Matern U. (2004) "Furanocoumarin biosynthesis in Ammi
majus L. Cloning of bergaptol O-methyltransferase." Eur J Biochem. 271(5), 932-40.
Abstract.
Plants belonging to the Apiaceae or Rutaceae accumulate methoxylated psoralens, such as
bergapten or xanthotoxin, as the final products of their furanocoumarin biosynthesis, and the rate of accumulation depends on environmental and other cues. Distinct O-methyltransferase
activities had been reported to methylate bergaptol to bergapten and xanthotoxol to  anthotoxin, from induced cell cultures of Ruta graveolens, Petroselinum crispum and Ammi majus. Bergaptol 5-O-methyltransferase (BMT) cDNA was cloned from dark-grown Ammi majus L. cells treated with a crude fungal elicitor. The translated polypeptide of 38.7 kDa, composed of 354 amino acids, revealed considerable sequence similarity to heterologous caffeic acid 3-Omethyltransferases (COMTs). For homologous comparison, COMT was cloned from A. majus
plants and shown to share 64% identity and about 79% similarity with the BMT sequence at the
polypeptide level. Functional expression of both enzymes in Escherichia coli revealed that the
BMT activity in the bacterial extracts was labile and rapidly lost on purification, whereas the
COMT activity remained stable. Furthermore, the recombinant AmBMT, which was most active in
potassium phosphate buffer of pH 8 at 42 degrees C, showed narrow substrate specificity for
bergaptol (Km SAM 6.5 micro m; Km Bergaptol 2.8 micro m) when assayed with a variety of
substrates, including xanthotoxol, while the AmCOMT accepted 5-hydroxyferulic acid, esculetin
and other substrates. Dark-grown A. majus cells expressed significant BMT activity which
nevertheless increased sevenfold within 8 h upon the addition of elicitor and reached a transient maximum at 8-11 h, whereas the COMT activity was rather low and did not respond to the elicitation. Complementary Northern blotting revealed that the BMT transcript abundance
increased to a maximum at 7 h, while only a weak constitutive signal was observed for the COMT transcript. The AmBMT sequence thus represents a novel database accession specific for the biosynthesis of psoralens.

Ammi visnaga oil Ammi visnaga (L.) Lam.
Cropwatch summary: [This entry is added for completeness: The coumarins in Ammi visnaga essential oil are mainly photochromones]. Ammi visnaga essential oil is produced in commercially, used in aromatherapy. Contains the spasmolytic furanochromone khellin & the pyranochromone visnagin, used to pigment skin via UV xposure in vitiligo treatment; photochemical cross-linkage to DNA disputed by photo-sensitised DNA cleavage sudy. (Chen & Kagan 1993).

Analysis for 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.

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
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-6ı,7ıdihydroxybergamottin antibody was obtained by immunizing rabbits with 6ı,7ıdihydroxybergamottin conjugated with bovine serum albumin using the N-succinimidyl ester
method. An enzyme marker was similarly prepared by coupling 6’,7’-dihydroxybergamottin with b-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 ytochrome P450 3A4 inhibitors in grapefruit juice.

Xie Y, Chen Y, Lin M, Wen J, Fan G, Wu Y. (2007) "High-performance liquid chromatographic
method for the determination and pharmacokinetic study of oxypeucedanin hydrate and byakangelicin after oral administration of Angelica dahurica extracts in mongrel dog plasma." J
Pharm Biomed Anal. 44(1), 166-72. Abstract. A high-performance liquid chromatographic method
was developed and validated for the determination and pharmacokinetic study of oxypeucedanin hydrate and byak-angelicin after oral administration of Angelica dahurica extracts in mongrel dog plasma. The coumarin components and the internal standard isopsoralen were extracted from plasma samples with the mixture of tert-butyl methyl ether and n-hexane (4:1, v/v).
Chromatographic separation was performed on a C(18) column (200 mm x 4.6mm, 5 microm)
with the mobile phase acetonitrile-methanol-water-acetic acid (20:15:65:2, v/v/v/v) at a flow-rate of 1.0 ml/min. Only the peak of oxypeucedanin hydrate and byak-angelicin could be detected in dog plasma after oral administration of ethanol extracts of A. dahurica mainly containing xanthotoxol, osthenol, imperatorin, oxypeucedanin hydrate and byakangelicin. The calibration curves of oxypeucedanin hydrate and byak-angelicin were linear over a range of 22.08-8830.00 and 6.08-2430.00 ng/ml in dog plasma, respectively. The quantification limit of oxypeucedanin hydrate and byak-angelicin in dog plasma was 22.08 and 6.08 ng/ml, respectively. The intra- and inter-day precision was less than 7.6% and 8.5% and the accuracy was from 91.9% to 106.1%.
The lowest absolute recoveries of oxypeucedanin hydrate and byak-angelicin were 85.7% and
87.0%, respectively. The method was successfully applied to the pharmacokinetic studies of
oxypeucedanin hydrate and byak-angelicin in dog plasma after oral administration of ethanol
extracts from A. dahurica.

Angelica oils. (A. archangelica spp. & subspp. only).
CAS n° 8015-64-3; EINECS-CAS n°: 84775-71-7
Cropwatch summary:
Phototoxicity: RIFM found Angelica root oil phototoxic: Opdyke D.L.J. (1975).
IFRA (2001) recommends 0.8% max.of Angelica root oil in leave-on products applied to skin then exposed to sunshine. However, the essential oil obtained by steam distillation of roots of A. archangelica was previously claimed free of furanocoumarins by EMEA (1996); root & seed essential oils were also claimed to be free of FC’s therefore not phototoxic by Leung (1996). RIFM originally found angelica seed oil non-photo-toxic (?): Opdyke (1974) through Tisserand &
Balacs (1995) p 116.

Composition: Harmala et al. (1992) identified the coumarins 2-angeloyl-3- isovaleryl vaginate, archangelicin, oxypeucedanin hydrate, bergapten, byakangelicin angelate, imperatorin, isoimperatorin, isopimpinellin, 8-[2-(3- methylbutroxy)-3-hydroxy-3-methylbutoxy]psoralen, osthol, ostruthol, oxypeucedanin, phellopterin, psoralen & xanthotoxin from a chloroform extract of the roots of Angelica archangelica L. ssp. archangelica.

Academic studies of lab. prepared root oils of Angelica archangelica L. ssp. archangelica var. sativa (Miller) Rikli reveal the presence of the angular furanocoumarins angelicin & archangelicin, as well as lesser amounts of linear furanocoumarins. Solvent extracts of Angelica spp. (often passed off as essential oils) contain angelicin, bergaptene, imperatorin, oxypeucedanin hydrate,
xanthotoxin and xanthotoxol.

Little reliable information on the FC contents of aromatic commodities from the seeds & roots of the various species & subspecies and geographic origins is available. Angelica species used to produce aromatic commodities include:
Angelica archangelica L. ssp. archangelica var. sativa (Miller): European oil.
A. sinensis (Oliv.) Diels. Chinese oil / extract.
A. koreana Maxim. Contains imperatorin, isoimperatorin, oxypeucedanin, isooxypeucedanin, & oxypeucedanin methanolate (Woo et al. 1983)

Exposure to Angelica furanocoumarins can occur via herbal medicine, & the EMEA considers that an exposure of 15 μg/day would not result in increased risk of photomutagenicity in consumers according to EMEA (2007).

RIFM give the following data “measured by the fragrance industry “ (??) for Angelica root oil (no botanical or geographical details given): psoralen up to 112 ppm, bergapten up to 78 ppm, angelicin 230 ppm; xanthotoxin, isopimpernellin & bergamottin were not detected (RIFM Fact Sheet No 3.).

References:
In het originele document. In verband met de ruimte heb ik een aantal referencies verwijderd. Origineel zal ik toesturen op verzoek - 92 pagina's.

Angelicin (isopsoralen).
CAS n°: 523-50-2.
Angular furanocoumarin. Angular furanocoumarins normally only produce DNAadducts, whereas linear furanocoumarins produce DNA-adducts and the potentially more toxic DNA-DNA interstrand cross links. Angelicin occurs to 230 ppm in angelica root oil & 430 ppm in rue oil (IFRA 2007). Angelicin is absent in citrus oils (IFRA 2007). There is limited evidence of carcinogenicity of angelicin to experimental animals in combination with UV radiation; inadequate evidence of
carcinogenicity to experimental animals in absence of UV radiation (IARC 1986).
No evaluation of carcinogenicity of angelicin could be made to humans (IARC 1986).

Aniseed oil. Pimpinella anisum L.
CAS n°: 8007-70-3; EINECS CAS No: 84775-45-1
Cropwatch summary: According to Leung (1996), plant tissues contain bergapten; however steam distilled seed oil not traditionally considered phototoxic.
Leung A.Y. & Foster S. Encyclopaedia of Common Natural Ingredients used in Food, Drugs &
Cosmetics 2nd edn. Wiley-Interscience pub. 1996.

Anti-carcinogenic / anti-genotoxic effects of furanocoumarins.
Imperatorin shows anti-carcinogenic effect against chemically induced cancer in a murine model (Prince et al. 2006), and, as does isopimpinellin, when administered in the diet (Kleiner et al. 2004). Kleiner et al. (2003) showed furanocoumarins are capable of inhibiting carcinogen-metabolising enzymes, & more specifically that bergamottin selectively inhibits tumor formation by benzo[a]pyrene, and that imperatorin & isopimpinellin block tumor initiation by both benzo[a]pyrene & 7,12-dimethylbenz[a]anthracene.

Bakkali F, Averbeck S, Averbeck D, Idaomar M. (2008) "Biological effects of essential oils - a
review." Food Chem Toxicol. 46(2),:446-75.
Abstract
Since the middle ages, essential oils have been widely used for bactericidal, virucidal, fungicidal, antiparasitical, insecticidal, medicinal and cosmetic applications, especially nowadays in pharmaceutical, sanitary, cosmetic, agricultural and food industries. Because of the mode of extraction, mostly by distillation from aromatic plants, they contain a variety of volatile molecules such as terpenes and terpenoids, phenolderived aromatic components and aliphatic components. In vitro physicochemical assays characterise most of them as antioxidants. However, recent work shows that in eukaryotic cells, essential oils can act as prooxidants affecting inner cell membranes and organelles such as mitochondria. Depending on type and concentration, they exhibit cytotoxic effects on living cells but are usually non-genotoxic. In some cases, changes in intracellular redox potential and mitochondrial dysfunction induced by essential oils can be associated with their capacity to exert antigenotoxic effects. These findings suggest that, at least in part, the encountered beneficial effects of essential oils are due to prooxidant effects on the cellular level.

Bakkali F., Averbeck S., Averbeck D., Zhiri A. & Idaomar M. (2005) "Cytotoxicity and gene
induction by some essential oils in the yeast Saccharomyces cerevisiae." Mutat Res. 585(1-2):1-
13.
Abstract.
In order to get an insight into the possible genotoxicity of essential oils (EOs) used
in traditional pharmacological applications we tested five different oils extracted from the
medicinal plants Origanum compactum, Coriandrum sativum, Artemisia herba alba,
Cinnamomum camphora (Ravintsara aromatica) and Helichrysum italicum (Calendula officinalis)
for genotoxic effects using the yeast Saccharomyces cerevisiae. Clear cytotoxic effects were
observed in the diploid yeast strain D7, with the cells being more sensitive to EOs in exponential than in stationary growth phase. The cytotoxicity decreased in the following order: Origanum compactum >Coriandrum sativum>Artemisia herba alba>Cinnamomum camphora>Helichrysum italicum. In the same order, all EOs, except that derived from Helichrysum italicum, clearly induced cytoplasmic petite mutations indicating damage to mitochondrial DNA. However, no nuclear genetic events such as point mutations or mitotic intragenic or intergenic recombination were induced. The capacity of EOs to induce nuclear DNA damage-responsive genes was tested using suitable Lac-Z fusion strains for RNR3 and RAD51, which are genes involved in DNA metabolism and DNA repair, respectively. At equitoxic doses, all EOs demonstrated significant gene induction, approximately the same as that caused by hydrogen peroxide, but much lower than that caused by methyl methanesulfonate (MMS). EOs affect mitochondrial structure and function and can stimulate the transcriptional expression of DNA damage-responsive genes. The induction of mitochondrial damage by EOs appears to be closely linked to overall cellular cytotoxicity and appears to mask the occurrence of nuclear genetic events. EO-induced cytotoxicity involves oxidative stress, as is evident from the protection observed in the presence of ROS inhibitors such as glutathione, catalase or the iron-chelating agent deferoxamine.

Bode C.W., Zager A. & Hansel W. (2005) "Photodynamic and photo-cross-linking potential of
bergamottin." Pharmazie. 60(1), 78-9.
Abstract.
Bergamottin (5-geranoxypsoralen) is a main component of bergamot and grapefruit oil. In order to investigate the photophysical and photochemical behaviour of bergamottin, absorption and fluorescence properties, production of singlet oxygen and superoxide radical anions and further cross-linking of DNA were studied.
Strong photochemical reactions were not observed.

Cai Y., Baer-Dubowska W., Ashwood-Smith M. & DiGiovanni J. (1997a) “Inhibitory effects of
naturally occurring coumarins on the metabolic activation of benzo(a)pyrene and 7,12-
dimethylbenz(a)anthracene in cultured mouse keratinocytes.” Carcinogen 18, 215-222.
Abstract.
Several naturally occurring coumarins to which humans are routinely exposed have been
previously found to be potent inhibitors and inactivators of cytochrome P450 (P450) 1A1-
mediated monooxygenase in both murine hepatic microsomes and in a reconstituted system
using purified human P450 1A1 [Cai et al. (1993) Chem. Res. Toxicol., 6, 872-879 and Cai et al.
(1996) Chem. Res. Toxicol., 9, 729-736]. In the present study, several of these coumarins were
investigated for their inhibitory effects on the metabolism and metabolic activation of
benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) in cultured mouse
keratinocytes. Initial analysis of B[a]P metabolism in cultured keratinocytes showed that
imperatorin, isoimperatorin, coriandrin, and bergamottin, at concentrations of 2 nM equal with
B[a]P, reduced the formation of water-soluble metabolites of B[a]P by 33% to 57%. Bergamottin
and coriandrin were the most potent inhibitors of the compounds examined. HPLC analysis of
organic solvent-soluble metabolites of B[a]P indicated that all the coumarins tested significantly
reduced the formation of individual B[a]P metabolites (including phenols, diols and tetraols).
However, the greatest effect was on the formation of B[a]P tetraols. Additional experiments
determined the ability of selected coumarins to block covalent binding of B[a]P and DMBA to DNA
in keratinocytes. Bergamottin preferentially inhibited the binding of B[a]P to DNA by 56%, while
coriandrin preferentially inhibited the binding of DMBA to DNA by 48%. Notably, analysis of
individual DNA adducts formed from B[a]P and DMBA indicated that both bergamottin and
coriandrin specifically inhibited the formation of anti diol-epoxide DNA adducts derived from both
hydrocarbons. The preferential inhibitory effect of bergamottin and coriandrin on the formation of anti diol-epoxide adducts derived from DMBA was further confirmed by separation of anti- and
syn-diol-epoxide-DNA adducts using immobilized boronate chromatography. The current study
demonstrates that certain naturally occurring coumarins inhibited metabolic activation of B[a]P
and DMBA in cultured mouse keratinocytes and specifically inhibited the formation of DNA
adducts derived from the anti diol-epoxide diastereomers from either hydrocarbon. The current
data also suggest that certain naturally occurring coumarins may possess anticarcinogenic
activity toward polycyclic aromatic hydrocarbons.

Cai Y., Kleiner H., Johnston D, Dubowski A., Bostic S., Ivie W. & DiGiovanni J. (1997b) “Effect of
naturally occurring coumarins on the formation of epidermal DNA adducts and skin tumors
induced by benzo(a)pyrene and 7,12-dimethylbenz(a)anthrecene in SENCAR mice.” Carcinogen
18, 1521-1527, 1997.
Abstract. Several naturally occurring coumarins previously found to be
potent inhibitors of mouse hepatic ethoxyresorufin-O-deethylase (EROD) and/or
pentoxyresorufin-O-dealkylase (PROD) were examined for their effects on formation of
benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) DNA adducts in mouse
epidermis, as well as, their effects on skin tumor initiation by these polycyclic aromatic
hydrocarbons (PAH).
Bergamottin, a potent inhibitor of hepatic EROD, given topically 5 min prior to an initiating dose of B[a]P, significantly decreased total covalent binding of B[a]P to DNA in a dose-dependent manner 24 h after treatment. A dose of 400 nmol bergamottin reduced covalent binding of B[a]P by 72%. Coriandrin, at a dose of 400 nmol also significantly reduced total covalent binding of B[a]P by 59%. In addition, formation of the major (+)anti-B[a]P-diol epoxide- N2-dGuo adduct was selectively reduced by both of these coumarins. In contrast, bergamottin and coriandrin did not significantly decrease covalent binding of DMBA to epidermal DNA at doses of either 400 nmol or 800 nmol. Imperatorin and isopimpinellin, which are more potent inhibitors of hepatic PROD activity, significantly reduced overall binding of DMBA to epidermal DNA by 67% and 52%, respectively, when applied at doses of 400 nmol. These two coumarins also inhibited B[a]P-DNA adduct formation at similar doses but to a lesser extent. Imperatorin at a dose of 400 nmol dramatically decreased formation of covalent DNA adducts derived from both the anti and syn diol epoxides of DMBA. Bergamottin was a potent inhibitor of tumor initiation by B[a]P while coriandrin was less effective in this regard. Imperatorin was an effective inhibitor of
skin tumor initiation by DMBA and also inhibited complete carcinogenesis by this PAH. At dose
levels higher than those effective against DMBA, imperatorin also inhibited tumor initiation by
B[a]P. The results demonstrate that several naturally occurring coumarins possess the ability to
block DNA adduct formation and tumor initiation by PAHs such as B[a]P and DMBA. The
mechanism for reduced DNA adduct formation and tumor initiation appears to involve inhibition of the P450s involved in the metabolic activation of these hydrocarbons. Finally, the differential
effects of certain coumarins on B[a]P vs DMBA DNA adduct formation and tumor initiation may be
useful for dissecting the role of specific cytochromes P450 in their metabolic activation.
Kim Y-K., Kim Y.S & Ryu S.Y. (2006) "Anti-proliferative effect of furanocoumarins from the root of
Angelica dahurica on cultured human tumor cell lines." Phytotherapy Research 21(3), 288-290
Abstract.
A bioassay-guided fractionation of the root extract of Angelica dahurica (Umbelliferae)
led to the isolation of six furanocoumarins as active ingredients responsible for the antitumoral
property. The hexane soluble part of the extract demonstrated a signicant inhibition on the
proliferation of cultured human tumor cells such as A549 (non small cell lung), SK-OV-3 (ovary),
SK-MEL-2 (melanoma), XF498 (central nervous system) and HCT-15 (colon) in vitro, whereas
the remaining water soluble part exhibited poor inhibition. Intensive investigation of the hexane
soluble part of the extract yielded six furanocoumarins, i.e. isoimperatorin, cnidicin, imperatorin,
oxypeucedanin, byakangelicol, oxypeucedanin hydrate, all of which exhibited a signicant
inhibition on cell proliferation in a dose-dependent manner.

Kleiner H.E., Vuimiri S.V., Miller L., Johnson Jr. W.H., Whitman C.P. & DiGivanni J. (2001) “Oral
administration of naturally occurring coumarins leads to altered phase I & phase II enzyme
activities & 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.

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.

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.

Abstract. The antimutagenicity of 9 kinds of chemical compounds (osthol , Bergaten, Isopimpinellin , Imperatorin , Xanthotoxol , Xnthotoxin , Thymine , Uracil and unknown one that need authenticating) was studied with the Ames test and the micronucleus and chromosomal aberretion assays in mice in vivo. These compounds were isolated f rom the water ext ract of Cnidium monnieri (L. ) Cuss in Chinese t raditional medicine. The results showed that osthol. , bergapten , isopimpinellin , imperatorin , xanthotoxol and xanthotoxin had inhibited activity of mutagenicity of aflatoxin B1 . And osthol , bergapten , isopimpinellin , imperatorin also had high activity in inhibiting the chromosomal aberrations and micronuclei that induced by
cyclophosphamid in vivo. The other three compounds ( Thymine , Uracil and unknown one that
need authenticating) showed no antimutagenicity.

Bergamot oil - cold pressed (Sicily, Ivory Coast) Citrus aurantium L. subsp.
bergamia (Risso et Poit.) Engl.
CAS n° : 8007-75-8 (or 900807-75-8); EINECS-CAS n° 89957-91-5.
Cropwatch summary:
Phototoxicity. A string of older studies (see BoDD website) show that bergamot oil-containing perfumes are phototoxic. It must be remembered however that bergamot oil concentrations in fragrances were likely to be higher during this older period; there have been restrictions on the concentrations deployed sincethen, & the use of so-called ‘furanocoumarin-free’ types with lower total furanocumarin concentrations, may have reduced the adverse reaction frequency. Photoxicity of bergamot oil was demonstrated on mouse skin when irradiated with UV light (Opdyke 1973). IFRA restricts bergamot oil expressed to 0.4% in fragrances not washed off skin, & exposed to sunshine.

On the other hand, bergamot oil is not cytotoxic or mutagenic in the absence of light (Dijoux et al. 2006) & reported acute phototoxic side effects from perfumes are rare, repeated exposure to sunlight after perfume application uncommon… no reports of increase in epithelial tumour formation on areas of skin habitually exposed to perfume (Dubertret et al. 1990 & Chouroulinkov et al. through Lawrence 1991).

Composition. RIFM give the following data “measured by the fragrance industry” (??) for bergamot oil (but no process or geographical source details given): psoralen 0-20 ppm, bergapten up to 2300 ppm, bergamottin up to 22,000 ppm; xanthotoxin, isopimpinellin, oxypeucedanin & angelicin not detected (RIFM Fact Sheet No 3.)

According to the SCCNFP 07403/05, ‘bergamot oil’ - type & origin not specified - contains 2.2% bergamottin, but is also notorious` for its bergapten content (0.3% within a total FC content of 3.0%: Forlott, unpublished data).

McHale & Sheridan (1989) found 1830 mg/100 g of bergamottin and 270 mg/100 g of bergapten in expressed bergamot oil. Duclos (2000) stated that the bergaptene content in bergamot oil (Italian) was 0.279% & that of bergamot oil Ivory Coast was 0.203-0.366%. Poiana et al. (1993) had determined the bergapten content of bergamot oil at 0.3-0.4%. Rouzet et al. (1980) identified in the nonvolatile fraction of lemon oil: bergamottin, 5-isopentenoxy-7-
methoxycoumarin, 8-geranoxypsoralen, byakangelicol, oxypeucedanine hydrate and byakangelitin, estimating their concentration to be 0.91%.

Mondello et al. (1992) analysed 128 genuine bergamot oils from Calabria (1992 production) & found bergamottin (1.02% to 2.75%) 5-geranyloxy-7-methoxycoumarin (0.08% to 0.22%), citropten (0.14% to 0.35%) & bergapten (0.11 to 0.32%). Citropen & bergapten content decreased from Jan. to Mar.

ijoux N., Guingand Y., Bourgeois C., Durand S., Fromageot C., Combe,C. & Ferret P.J., (2006)
"Assessment of the phototoxic hazard of some essential oils using modified 3T3 neutral red
uptake assay." Toxicol. in Vitro 20, 480–489. Abstract. When substances are developed in the
aim to be a constituent of personal care products, and to be applied on the skin, it is necessary to carry out an assessment of potential phototoxic hazard. Phototoxicity is skin reaction caused by concurrent topical or systemic exposure to specific molecule and ultraviolet radiation. Most
phototoxic compounds absorb energy particularly from UVA light leading to the generation of
activated derivatives which can induce cellular damage. This type of adverse cutaneous response can be reproduced in vitro using different models of phototoxicity such as the validated 3T3 Neutral Red Uptake (NRU) phototoxicity assay. In the present study we utilised two different cell lines (the murine fibroblastic cell line 3T3 and the rabbit cornea derived cell line SIRC) to compare the photo-irritation potential of a strong phototoxic compound, hlorpromazine, to a weaker composite, such as 8-methoxypsoralen and Bergamot oil. After comparison of the
different systems, five other essential oils were tested with both cell lines. Cellular damage was
evaluated by the NRU cytotoxicity test or by MTT conversion test.
Dubertret L., Morlière P., Averbeck D. & Young A.R. (1990) “The photochemistry and photobiology of bergamot oil as a perfume ingredient: an overview.” J Photochem Photobiol B.
7(2-4), 362-5

Dubertret L., Serraf-Tircazes D., Jeanmougin M., Morliere P., Averbeck D. & Young A.R. (1990)
“Phototoxic properties of perfumes containing bergamot oil on human skin. Photoprotective effect of UVA and UVA substances.” J. Photochem. Photobiol. 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.

Kaddu S., Kerl H., & Wolf P. (2001) “Accidental bullous phototoxic reactions to bergamot
aromatherapy oil.” J. Am. Acad Dermatol 45(3), 458-461. Abstract. Oil of bergamot is an extract
from the rind of bergamot orange (Citrus aurantium ssp bergamia) that has a pleasant, refreshing scent; until a few years ago it had been widely used as an ingredient in cosmetics but was restricted or banned in most countries because of certain adverse effects. More recently, oil of bergamot preparations have been gaining renewed popularity in aromatherapy. Oil of bergamot possesses photosensitive and melanogenic properties because of the presence of furocoumarins, primarily bergapten (5-methoxypsoralen [5-MOP]). However, 5-MOP is also
potentially phototoxic and photomutagenic. Despite its increasing application, there are only a few recent reports of phototoxic reactions to bergamot aromatherapy oil. We describe two patients with localized and disseminated bullous phototoxic skin reactions developing within 48 to 72 hours after exposure to bergamot aromatherapy oil and subsequent ultraviolet exposure. One patient (case 2) had no history of direct contact with aromatherapy oil but developed bullous skin lesions after exposure to aerosolized (evaporated) aromatherapy oil in a sauna and subsequent UVA radiation in a tanning salon. This report highlights the potential health hazard related to the increasing use of psoralen-containing aromatherapy oils.

Mondello L., d'Alcontres I.S., Del Duce R., & Francesco Crispo (1993) "On the genuineness of
citrus essential oils. Part XL. The composition of the coumarins and psoralens of Calabrian
bergamot essential oil (Citrus bergamia Risso)." Flav & Frag J. 8(1), 17-24. Abstract. The content of bergamottin, 5-geranyloxy-7-methoxycoumarin, citropten and bergapten in Calabrian bergamot essential oil was determined by HPLC. 128 genuine bergamot essential oils, cold-pressed during the year 1992, were analysed; for each sample the date and area of production of the fruits is known. The percentage of bergamottin ranges between 1.02% and 2.75%. 5-geranyloxy-7-methoxycoumarin between 0.08 and 0.22%, citropten between 0.14% and 0.35%, bergapten between 0.11 and 0.32%. The mean content of bergamottin and 5-geranyloxy-7-methoxycoumarin did not vary during the production period, while that of citropten and bergapten decreased considerably from January to March. There does not seem to be any evident correlation between the production areas of the fruits and the coumarin composition. The presence of some peaks absent in genuine bergamot essential oils, has been detected in three bergamot oils, available on the market, probably because of lime oil addition.

Abstract. Bases for the elaboration of a standardized protocol are proposed for studying
phototoxic effects of skin tanning preparations containing photosensitizing agents. The
experimental procedure includes in vivo phototoxicity tests, evaluation of the photogenotoxic risk and determination of the photosensitizer concentration in plasma after topical application. This procedure was carried out with tanning preparations containing a well-known photosensitizer, 5- methoxypsoralen, as a component of bergamot oil. The whole study has been performed using topical application of the commercial suntan product, i.e. containing the sunscreens and all other components. Whereas the exposure to solar simulated radiation never triggered any phototoxic response, a photosensitizing effect was observed for skin type I volunteers exposed to high doses of ultraviolet A. The transepidermal penetration resulted in a 5-methoxypsoralen concentration of 1-4 ng/ml in the suction blister fluid. The photogenotoxicity of this suction blister fluid containing 5-methoxypsoralen and also other ingredients of the tanning preparation was assayed on yeast cells and was found to be rather low. 5-Methoxypsoralen was also detected in plasma after repeated applications but at low concentrations (about 1 ng/ml) which do not present a potential risk for systemic ocular effects.

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 xposure defers the time-to-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.

Bergamot oil distilled.
Cropwatch summary: Rectified oils may contain approx. 300 ppm bergaptene according to one Italian manufacturer; however up to now distilled oils have generally been assumed to be non-phototoxic e.g. by S. Arctander (through Opdyke 1973). See section below undergo Dugo (1999).

Bergamot oil “FCF” (“FuranoCoumarin-Free”).
Cropwatch summary Bergamot FCF oils contain from zero to 9-10 ppm bergaptene when pure (Cropwatch consensus of several manufacturers, 2007).
However much of the commercially available bergamot oil FCF is adulterated, lowering the FCF content further. Higher boiling fractions of distilled bergamot oil (which contain valuable odour compounds) may be treated by column chromatography, chemo-absorbtion techniques or by solvent extraction to reduce overall FCF content, and are added back to lower boiling fractions (thanks to D. Joulain & staff from Italian citrus producers who don’t want to be named, for this
information). Dugo et al. (1999) gave the following information for
‘Furanocoumarin Free bergamot oil’:
          (a) sodium hydroxide treated bergamot oil: bergapten 0-91 ppm; bergamottin 11,726-16,250 ppm, 5-geranyloxy-7-methylcoumarin 1539-1975 ppm.
          (b) distilled bergamot oil: bergapten 0-41 ppm; bergamottin 0-3017 ppm; 5-
geranyloxy-7-methylcoumarin 0-349 ppm.

Bergamottin (5-geranoxypsoralen).
CAS n°: 7380-40-7; EC n°: 200-782-5
Stability. Bergamottin is not particularly stable & tends to decompose during GC run conditions, making GC estimation unreliable. Morliere et al. (1990) found that not only was bergamottin responcible for “about two-thirds of the absorption of UVA and UVB light by bergamot oil,” but that bergamottin in bergamot oil was unstable to UV light in 80% ethanol. Under mildly acidic conditions bergamottin can be converted to bergaptol (Stanley & Vannier 1957).
Ocurrence. Found at 1253.37 ppm by UV-DAD in cold-expressed Californian  lemon oil Citrus limonum (L.) (N. L. Burman), at 428.6 ppm in cold-pressed Israeli grapefruit oil Citrus paradisis Macf syn. Citrus decumana L., and at 111.6 ppm in cold-pressed bitter orange oil Citrus aurantium L. subsp. amara. (Frerot & Decorzant 2004). The SCCP Opinion SCCNFP/0740/03 quoted the bergamottin concentration at 2.2% in bergamot oil & 2.5% in cold-pressed lime oil. Alkaline treatment of expressed bergamot oil to remove bergapten still leaves bergamottin
as major furanocumarin (Frerot & Decorzant 2004).
Phototoxicity. The lack of interaction of bergamottin with DNA has been noted (Aubin et. al. 1994; Bode et al. 2005; Morliere et al. 1991). The SCCNFP Opinion 0740/03 accordingly notes that bergamottin inhibits cytochrome P450 CYP3A and CYP1A1/2 enzymatic activities and induction of the corresponding proteins and mRNAs (see Cai et al 1997a; Cai et al 1997b). The SCCNFP Opinion SCCPNFP/0740/03 concluded there was insufficient information (as submitted by EFFA) on bertgamottin’s photo-toxic potential to come to an opinion regarding its
safety in cosmetic products. RIFM subsequently commissioned David Kirkland of Covance Labs. to provide further studies where we are told (RIFM 2007, 2008) that that “bergamottin induced structural chromosome aberrations in the presence of UV light, indicating a photoclastogenic response” - however full experimental details are not in the public domain.

Abstract. 5-geranoxypsoralen (5-GOP), commonly called bergamottin, is a highly photoreactive
psoralen, which in contrast to most furocoumarins, does not strongly interact with DNA. 5-GOP
gives the opportunity to study, in a more selective way, the mechanisms of phototoxic and
immunological activities induced by psoralen and UVA radiation. We investigated the effects of
repetitive treatments with 5-GOP plus UVA radiation (320-400 nm) on the number of ATPase+
epidermal Langerhans cells and on the induction of photoreactivity. These effects were compared with those of 8-methoxypsoralen (8-MOP) or 5-methoxypsoralen (5-MOP) plus UVA radiation and UVA radiation alone. C3H/HeN mice were treated topically with the psoralen three times/week for 4 consecutive weeks followed each time by 1 J/cm2 of UVA radiation. At the end of the treatment, mice treated with 8-MOP or 5-MOP plus UVA radiation exhibited severe gross phototoxicity and nearly total depletion of ATPase-stained Langerhans cells. Both treatments produced severe morphological alterations of Langerhans cells. No gross but a microscopic phototoxic effect was observed after 5-GOP plus UVA radiation treatment, while the number of ATPase+ Langerhans cells was also greatly reduced. Interestingly the latter treatment induced no morphological lterations of the remaining Langerhans cells in contrast to treatment with 8-MOP or 5-MOP plus VA radiation. We conclude that phototoxicity and decrease in the number of ATPase-stained pidermal immune cells observed after treatment with 5-GOP plus UVA radiation are not related o the DNA binding activity of the psoralen.

Bode C.W., Zager A. &Hansel W. (2005) "Photodynamic and photo-cross-linking potential of
bergamottin." Pharmazie. 60(1), 78-9. Abstract. Bergamottin (5-geranoxypsoralen) is a main
component of bergamot and grapefruit oil. In order to investigate the photophysical and
photochemical behaviour of bergamottin, absorption and fluorescence properties, production of
singlet oxygen and superoxide radical anions and further cross-linking of DNA were studied.
Strong photochemical reactions were not observed.

Cai Y., Baer-Dubowska W., Ashwood-Smith M., DiGiovanni J. (1997a) “Inhibitory effects of
naturally occurring coumarins on the metabolic activation of benzo(a)pyrene and 7,12-
dimethylbenz(a)anthracene in cultured mouse keratinocytes.” Carcinogen 18, 215-222. Abstract.
Several naturally occurring coumarins to which humans are routinely exposed have been
previously found to be potent inhibitors and inactivators of cytochrome P450 (P450) 1A1-
mediated monooxygenase in both murine hepatic microsomes and in a reconstituted system
using purified human P450 1A1 [Cai et al. (1993) Chem. Res. Toxicol., 6, 872-879 and Cai et al.
(1996) Chem. Res. Toxicol., 9, 729-736]. In the present study, several of these coumarins were
investigated for their inhibitory effects on the metabolism and metabolic activation of
benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) in cultured mouse
keratinocytes. Initial analysis of B[a]P metabolism in cultured keratinocytes showed that
imperatorin, isoimperatorin, coriandrin, and bergamottin, at concentrations of 2 nM equal with
B[a]P, reduced the formation of water-soluble metabolites of B[a]P by 33% to 57%. Bergamottin
and coriandrin were the most potent inhibitors of the compounds examined. HPLC analysis of
organic solvent-soluble metabolites of B[a]P indicated that all the coumarins tested significantly
reduced the formation of individual B[a]P metabolites (including phenols, diols and tetraols).
However, the greatest effect was on the formation of B[a]P tetraols. Additional experiments
determined the ability of selected coumarins to block covalent binding of B[a]P and DMBA to DNA
in keratinocytes. Bergamottin preferentially inhibited the binding of B[a]P to DNA by 56%, while
coriandrin preferentially inhibited the binding of DMBA to DNA by 48%. Notably, analysis of
individual DNA adducts formed from B[a]P and DMBA indicated that both bergamottin and
coriandrin specifically inhibited the formation of anti diol-epoxide DNA adducts derived from both
hydrocarbons. The preferential inhibitory effect of bergamottin and coriandrin on the formation of nti diol-epoxide adducts derived from DMBA was further confirmed by separation of anti- and
syn-diol-epoxide-DNA adducts using immobilized boronate chromatography. The current study
demonstrates that certain naturally occurring coumarins inhibited metabolic activation of B[a]P
and DMBA in cultured mouse keratinocytes and specifically inhibited the formation of DNA
adducts derived from the anti diol-epoxide diastereomers from either hydrocarbon. The current
data also suggest that certain naturally occurring coumarins may possess anticarcinogenic
activity toward polycyclic aromatic hydrocarbons.

Abstract. Several naturally occurring coumarins previously found to be otent inhibitors of mouse hepatic ethoxyresorufin-O-deethylase (EROD) and/or entoxyresorufin-O-dealkylase (PROD) were examined for their effects on formation of enzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) DNA adducts in mouse epidermis as well as, their effects on skin tumor initiation by these polycyclic aromatic hydrocarbons(PAH). Bergamottin, a potent inhibitor of hepatic EROD, given topically 5 min prior of an initiating dose of B[a]P, significantly decreased total covalent binding of B[a]P to DNA in a dose-dependent manner 24 h after treatment. A dose of 400 nmol bergamottin reduced covalent binding of B[a]P by 72%. Coriandrin, at a dose of 400 nmol also significantly reduced total covalent binding of B[a]P by 59%. In addition, formation of the major (+)anti-B[a]P-diol epoxide- 2-dGuo adduct was selectively reduced by both of these coumarins. In contrast, bergamottin and coriandrin did not significantly decrease covalent binding of DMBA to epidermal DNA at doses of either 400 nmol or 800 nmol.

Imperatorin and isopimpinellin, which are more potent inhibitors of hepatic PROD activity, significantly reduced overall binding of DMBA to epidermal DNA by 67% and 52%, respectively, when applied at doses of 400 nmol.
These two coumarins  also inhibited B[a]P-DNA adduct formation at similar doses but to a lesser extent. Imperatorin at a  dose of 400 nmol dramatically decreased formation of covalent DNA adducts derived from both the anti and syn diol epoxides of DMBA. Bergamottin was a potent inhibitor of tumor initiation by  B[a]P while coriandrin was less effective in this regard. Imperatorin was an effective inhibitor of skin tumor initiation by DMBA and also inhibited complete carcinogenesis by this PAH. At dose  levels higher than those effective against DMBA, imperatorin also inhibited tumor initiation by B[a]P. The results demonstrate that several naturally occurring coumarins possess the ability to  block DNA adduct formation and tumor initiation by PAHs such as B[a]P and DMBA. The  mechanism for reduced DNA adduct formation and tumor initiation appears to involve inhibition of  the P450s involved in the metabolic activation of these hydrocarbons. Finally, the differential effects of certain coumarins on B[a]P vs DMBA DNA adduct formation and tumor initiation may be useful for dissecting the role of specific cytochromes P450 in their metabolic activation.

Abstract. Bergamottin, which accounts for about two-thirds of the  absorption of UVA and UVB light by bergamot oil, is shown to be fairly unstable on UV irradiation  of solutions of bergamot oil (in ethanol-water, 80:20 (w/w)). Bergamottin photodegradation is  partly  inhibited by        molecular oxygen and also by a cinnamate sunscreen acting as a triplet excited  state quencher. On UV irradiation of bergamot oil, type II photodynamic properties, i.e. singlet oxygen roduction, are observed, which can be mainly attributed to the excitation of bergamottin  by light. There for bergamottin can be considered as a potential photosensitizer in the photobiological activity of bergamot oil.

Morliere P., Bazin M., Dubertret L., Santus R., Sa E Melo T, Huppe G., Haigle J, Forlot P &
Bernard A. (1991) "Photoreactivity of 5-geranoxypsoralen and lack of photoreaction with DNA".
Photochem Photobiol. 53(1),13-9. Abstract. 5-Geranoxypsoralen, commonly called bergamottin, a major furocoumarin contained in bergamot oil, is reported in vitro as a highly photoreactive
psoralen. In ethanol, it exhibits quite a high triplet state quantum yield (approximately 0.37). The triplet state is involved in subsequent photochemistry which depends on the initial concentration and on the presence of oxygen. In contrast to most psoralens, absorption and fluorescence data suggest that 5-geranoxypsoralen does not interact with DNA in the dark. No UVA-induced interstrand cross-links in DNA were shown.

Naar furocoumarine vervolg 1