Biological effects of essential oils--a review.

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, phenol-derived 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.

Antigenotoxic effects of three essential oils in diploid yeast (Saccharomyces cerevisiae) after treatments with UVC radiation, 8-MOP plus UVA and MMS.

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

Cytotoxicity and gene induction by some essential oils in the yeast Saccharomyces cerevisiae.

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.

DNA photodamage, repair, gene induction and genotoxicity following exposures to 254 nm UV and 8-methoxypsoralen plus UVA in a eukaryotic cell system.

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

Enzymatic recognition and biological effects of photodynamic damage induced in DNA by 1,6-dioxapyrene plus UVA.

The specific recognition of DNA modifications by repair endonucleases was used to characterize DNA damage induced by 1,6-dioxapyrene (1,6-DP) in the presence of ultraviolet light at 365 nm (UVA) in the plasmid YEplac181. Under cell free conditions, 1,6-DP plus UVA generated lesions are recognized by the UvrABC endonuclease, the proteins Nth, Nfo and Fpg. The number of UvrABC sensitive sites was at least ten-fold higher than that of Fpg or Nth sensitive sites. Moreover, 1,6-DP plus UVA generated single-strand breaks which are the second most frequent lesions. To investigate the biological effect of DNA damage, YEplac181 DNA was treated with 1,6-DP plus UVA and transformed into Escherichia coli or Saccharomyces cerevisiae. In Escherichia coli, the transformation efficiency of 1,6-DP plus UVA treated DNA was greatly reduced in the uvrA mutant compared to that in the wild-type strain. However, the transforming efficiency was not affected in Fpg-deficient strains. In Saccharomyces cerevisiae, the transformation efficiency of 1,6-DP plus UVA treated YEplac181 was greatly reduced in the rad14::URA3 strain. The photobiological effect of 1,6-DP plus UVA was also analysed in haploid yeast strains of various repair capacities. The results show that the yeast strain defective in the nucleotide excision repair pathway (rad14::URA3) is hypersensitive to 1,6-DP plus UVA treatment as compared to the parental wild-type strain. It is confirmed that the lethal effect of 1,6-DP plus UVA on wild-type yeast is strongly oxygen dependent, whereas the survival of the rad14::URA3 mutant only exhibits a minor oxygen dependence. To conclude, our data show that the photodynamic DNA lesions induced by 1,6-DP plus UVA can be recognized and repaired in pro- and eukaryotic cells by the nucleotide excision repair pathway.

Induction of the genes RAD54 and RNR2 by various DNA damaging agents in Saccharomyces cerevisiae.

The relationship between the induction of the genes RAD54 and RNR2 and the induction and repair of specific DNA lesions was studied in the yeast Saccharomyces cerevisiae using Rad54-lacZ and RNR2-lacZ fusion strains. Gene induction was followed by measuring beta-galactosidase activity. At comparable levels of furocoumarin-DNA photoadducts, RAD54 was more effectively induced by bifunctional than by monofunctional furocoumarins indicating that mixtures of monoadducts (MA) and interstrand cross-links (CL) provide a stronger inducing signal than MA. RNR2 induction kinetics were measured in relation to cell growth and survival responses after treatment with the furocoumarins 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP), 3-carbethoxypsoralen (3-CPs), 7-methyl-pyrido[3,4-c]psoralen (MePyPs) and 4,4',6-trimethylangelicin (TMA), benzo[a]pyrene (B(a)P and 1,6-dioxapyrene (1,6-DP) plus UVA, 254 nm UV radiation and cobalt-60 gamma-radiation. Induction of RNR2 took place during the DNA repair period before resumption of cell growth and clearly increased with increasing equitoxic dose levels. Treatments with furocoumarin plus 365 nm radiation (UVA) and 254 nm (UV) radiation were effective inducers whereas gene induction was relatively weak after gamma-radiation and absent after the induction of oxidative damage by B(a)P and 1,6-DP and UVA. The results suggest that it is the specific processing of different DNA lesions that determines the potency of the induction signal. Apparently, DNA lesions such as CL, and probably also closely located MA or pyrimidine dimers in opposite DNA strands involving the formation of double-strand breaks as repair intermediates, are most effective inducers.

Biophysical and biological properties of newly synthesized dioxinocoumarin derivatives. II. Dark and photoinduced effects on T7 phage, yeast and HeLa cells.

The dioxinocoumarin derivatives 5H-[2]benzopyrano-[3,4-g][1,4]benzodioxin-5-one (I), 5H-[2]benzopyrano-[3,4-g][2,3]-dihydro-[1,4]benzodioxin-5-on e II, 6H-[2]benzopyrano[3,4-f]-1,4-benzodioxin-6-one (III) and 6H-[2]benzopyrano[3,4-f]-2,3-dihydro-1,4-benzodioxin-6-one (IV) were synthesized. Their biological effect was studied in the presence and absence of UVA radiation, and compared with that of 8-methoxypsoralen (8-MOP) and angelicin derivatives on T7 phage, diploid yeast (Saccharomyces cerevisiae) and HeLa cells. The photobiological activities of compounds I and III were stronger than that of 8-MOP in phage inactivation and DNA synthesis inhibition in HeLa cells, whereas compounds II and IV, with a saturated dioxin ring, showed very poor activity. The photosensitizing activity of dioxinocoumarins on phage inactivation decreased by a factor of two to three in the absence of oxygen. Treatments with compound I and UVA in the presence of oxygen modified the helical structure and stability of phage DNA and proteins. Compounds I and II were more active than IV for photoinduced cell killing in yeast, although always less active than 8-MOP. At comparable photocytotoxic levels, compounds I and III were as strong inducers of cytoplasmic "petite" mutants in yeast as angelicin, suggesting a possible monofunctional mode of action with cellular DNA.

Genotoxicity of bergapten and bergamot oil in Saccharomyces cerevisiae.

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 light-absorbing 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.

Lethal and mutagenic effects photoinduced in haploid yeast (Saccharomyces cerevisiae) by two new monofunctional pyridopsoralens compared to 3-carbethoxypsoralen and 8-methoxypsoralen.

The photobiological effects of two monofunctional pyridopsoralens (PPs), pyrido[3,4-c]psoralen and pyrido[3,4-c]-7-methylpsoralen were studied and compared to those of 3-carbethoxypsoralen (3-CPs) and 8-methoxypsoralen (8-MOP) in a haploid wild-type strain of yeast (Saccharomyces cerevisiae). The capacity of PPs to photoinduce lethal effects in the presence of 365-nm radiation was not only higher than that of the monofunctional compound 3-CPs, but also higher than that of the bifunctional compound 8-MOP. This activity was apparently independent of oxygen, and it was found that it was probably due to the induction of monoadducts in DNA. A high effectiveness of PPs on the induction of cytoplasmic 'petite' mutations was observed suggesting a high photoaffinity towards mitochondrial DNA. In contrast to 8-MOP, the strong cell killing activity of PPs was not accompanied by a strong inducing effect on nuclear mutations (HIS+ reversions or canR forward mutations). For these endpoints, PPs were less effective per unit dose of 365-nm radiation and also less efficient per viable cell than 8-MOP. From this, it appears that the lesions photoinduced by the former compounds show a more lethal than (nuclear) mutagenic potential. Furthermore, the fact that PPs were even less mutagenic (nuclear) per viable cell than the monofunctional compound 3-CPs suggests that the activity of these agents may differ in frequency and nature of lesions induced. The photobiological activity of PPs in haploid yeast appears to be in line with the recent proposition for their use in photochemotherapy.

Photobiological properties of furothiocoumarins in Saccharomyces cerevisiae.

Nine furothiocoumarins corresponding to psoralen, 8-methylpsoralen, 3-carbethoxy-8-methylpsoralen, 8-methoxypsoralen, pseudopsoralen, angelicin, isopseudopsoralen, allopsoralen and pseudoisopsoralen were synthesized by treating furocoumarins with phosphorus pentasulfide. Photobiological studies on haploid yeast cells (Saccharomyces cerevisiae) revealed that the furothiocoumarins exert some photoactivity on cell survival and the induction of mitochondrial damage. In most cases, the furothiocoumarins were less active than their furocoumarinic counterparts and exhibited a preference for a monofunctional type of action. A certain photochemotherapeutic activity can be suggested.

The metabolism of 8-methoxypsoralen by Saccharomyces cerevisiae. Evidence for an inducing effect of ethanol.

The metabolism of 8-methoxypsoralen (8-MOP) by the yeast Saccharomyces cerevisiae has been investigated in order to determine if this cell system could provide a simple model to study the metabolism of new photosensitizing drugs in vitro. 8-MOP was found to be rapidly metabolized by S. cerevisiae in non growing conditions. A metabolite was detected which exhibits a structure similar to that of a metabolite previously isolated in dog and man. Ethanol showed a strong inducing effect on 8-MOP metabolization.

Mutagenic activity of three monofunctional and three bifunctional furocoumarins in yeast (Saccharomyces cerevisiae).

The mutagenic activity of photoadditions induced by 4,5'-dimethylangelicin (4,5'-DMA), 3-carbethoxypsoralen (3-CPs), angelicin, 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP) and 4,5',8-trimethylpsoralen (TMP), was studied in haploid yeast cells at the nuclear and cytoplasmic levels. With regard to the induction of cell killing 4,5'-DMA and 3-CPs were about 5 to 6 times more active than 8-MOP probably due to an efficient repair of 4,5'-DMA or 3'CPs induced monoadditions in DNA. 4,5'-DMA was about 5-fold less active than angelicin, reflecting the different photoreactivities of the compounds towards DNA. In accord with its monofunctional activity, 4,5'-DMA was less efficient on the induction of nuclear reversions but more efficient on the induction of cytoplasmic "petite" mutants per viable cell than 8-MOP and the other bifunctional compounds. In contrast to 3-CPs and angelicin, the reversion induction by 4,5'-DMA followed a 2 hit kinetics. For the induction of CanR forward mutants per viable cell 4,5'-DMA was more efficient than 3-CPs and approached the activity of 8-MOP; angelicin, 5-MOP and TMP were slightly more efficient than 8-MOP, whereas 3-CPs was clearly less efficient. Thus, the monofunctional furocoumarins exert different genotoxic effects when compared on a survival basis. Cell killing effects in the presence and in the absence of oxygen were also evaluated.

Dose-rate effects of 8-methoxypsoralen plus 365-NM irradiation on cell killing in Saccharomyces cerevisiae.

Tow types of dose-rate effect that alter the survival response of haploid yeast cells to 8-methoxypsoralen (8-MOP) plus treatment with irradiation at 365 nm were studied. (1) When the concentration of 8-MOP was varied between 9.2 X 10(-5) and 2.3 X 10(-8) M and the dose rate of 365-nm irradiation kept constant, the efficiency of the irradiation for killing increased relatively to that of 8-MOP whe the concentration of 8-MOP decreased. This indicated that there was no strict reciprocity between radiation dose and concentration of drug. (2) When the dose rate of radiation was varied between 0.66 X 10(3) and 108 X 10(3) J m-2 h-1 and the concentration of 8-MOP was kept constant, the survival of wild-type cells increased strikingly at low dose rates of radiation as compared with high dose rates. Cells responded more to changes at low dose rates than to equal changes a high dose rates. The high resistance of wild-type cells to 8-MOP plus radiation delivered at low dose rates absent from rad 1-3 cells defective in excision-repair. This suggests that the dose-rate effect seen in wild-type cells depended at least in part on an active excision-repair function. At low dose rates of radiation, the shoulder of the survival curve for rad1-3 cells, i.e. the ability to accumulate sub-lethal damage, was increased by a factor of about 2 when compared with that seen at a high dose rate. Thus it is likely that at low dose rates a repair function other than excision-resynthesis may operate in rad1-3 cells.