Critical assessment of the endocrine potential of Linalool and Linalyl acetate: proactive testing strategy assessing estrogenic and androgenic activity of Lavender oil main components.

The acyclic linear monoterpenes Linalool (Lin) and Linalyl acetate (LinAc) occur in nature as major constituents of various essential oils such as lavender oils. A potential endocrine activity of these compounds was discussed in literature including premature thelarche and prepubertal gynecomastia due to lavender product use. This study aims to follow-up on these critical findings reported by testing Lin and LinAc in several studies in line with current guidance and regulatory framework. No relevant anti-/ER and AR-mediated activity was observed in recombinant yeast cell-based screening tests and guideline reporter gene in vitro assays in mammalian cells. Findings in the screening test suggested an anti-androgenic activity, which could not be confirmed in the respective mammalian cell guideline assay. Mechanistic guideline in vivo studies (Uterotrophic and Hershberger assays) with Lin did not show significant dose related changes in estrogen or androgen sensitive organ weights and a guideline reproductive toxicity screening study did not reveal evident effects on sex steroid hormone sensitive organ weights, associated histopathological findings and altered sperm parameters. Estrous cycling and mating/fertility indices were not affected and no evident Lin-related steroid hormone dependent effects were found in the offspring. Overall, the initial concerns from literature were not confirmed. Findings in the yeast screening test were aberrant from follow-up guideline in vitro and in vivo studies, which underlines the need to apply careful interpretation of single in vitro test results to support a respective line of evidence and to establish a biologically plausible link to an adverse outcome.

Genetic architecture of the toxicological response to eucalyptol and citronellal in Drosophila melanogaster.

The excessive and indiscriminate use of synthetic insecticides has led to environmental pollution, wildlife destruction, and adverse effects on human health, while simultaneously giving rise to resistance in insect pest populations. This adaptive trait is expressed through various mechanisms, such as changes in the cuticle, heightened activities of detoxifying enzymes, and alterations in the sites of action that reduce their affinity for insecticides. In this context, we associate variation in toxicological response with genomic variation, to identify genetic polymorphisms underlying the different steps of the insect (genotype)-response (phenotype)-insecticide (environment) interaction. Under this framework, our objective was to investigate the genetic factors involved in the toxicological response of D. melanogaster lines when exposed to citronellal and eucalyptol vapors (monoterpenes of plant origin). We quantified KT50 in adult males, representing the time necessary for half of the exposed individuals to be turned upside down (unable to walk or fly). Since the genomes of all lines used are completely sequenced, we perform a Genome Wide Association Study to analyze the genetic underpinnings of the toxicological response. Our investigation enabled the identification of 656 genetic polymorphisms and 316 candidate genes responsible for the overall phenotypic variation. Among these, 162 candidate genes (77.1%) exhibited specificity to citronellal, 45 (21.4%) were specific to eucalyptol, and 3 candidate genes (1.5%) namely CG34345, robo2, and Ac13E, were implicated in the variation for both monoterpenes. These suggest a widespread adaptability in the response to insecticides, encompassing genes influenced by monoterpenes and those orchestrating resistance to the toxicity of these compounds.

Toxicity of Selected Monoterpenes and Essential Oils Rich in These Compounds.

Monoterpenes make up the largest group of plant secondary metabolites. They can be found in numerous plants, among others, the Lamiaceae family. The compounds demonstrate antioxidative, antibacterial, sedative and anti-inflammatory activity, hence, they are often employed in medicine and pharmaceuticals. Additionally, their fragrant character is often made use of, notably in the food and cosmetic industries. Nevertheless, long-lasting studies have revealed their toxic properties. This fact has led to a detailed analysis of the compounds towards their side effects on the human organism. Although most are safe for human food and medical applications, there are monoterpene compounds that, in certain amounts or under particular circumstances (e.g., pregnancy), can cause serious disorders. The presented review characterises in vitro and in vivo, the toxic character of selected monoterpenes (α-terpinene, camphor, citral, limonene, pulegone, thujone), as well as that of their original plant sources and their essential oils. The selected monoterpenes reveal various toxic properties among which are embryotoxic, neurotoxic, allergenic and genotoxic. It is also known that the essential oils of popular plants can also reveal toxic characteristics that many people are unaware of.

Hinokitiol Exhibits Antitumor Properties through Induction of ROS-Mediated Apoptosis and p53-Driven Cell-Cycle Arrest in Endometrial Cancer Cell Lines (Ishikawa, HEC-1A, KLE).

Hinokitiol is a natural tropolone derivative that is present in the heartwood of cupressaceous plants, and has been extensively investigated for its anti-inflammatory, antioxidant, and antitumor properties in the context of various diseases. To date, the effects of hinokitiol on endometrial cancer (EC) has not been explored. The purpose of our study was to investigate the anti-proliferative effects of hinokitiol on EC cells. Cell viability was determined with an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and the quantification of apoptosis and reactive oxygen species (ROSs) was performed by using flow cytometry, while protein expression was measured with the Western blotting technique. Hinokitiol significantly suppressed cell proliferation through the inhibition of the expression of cell-cycle mediators, such as cyclin D1 and cyclin-dependent kinase 4 (CDK4), as well as the induction of the tumor suppressor protein p53. In addition, hinokitiol increased the number of apoptotic cells and increased the protein expression of cleaved-poly-ADP-ribose polymerase (PARP) and active cleaved-caspase-3, as well as the ratio of Bcl-2-associated X protein (Bax) to B-cell lymphoma 2 (Bcl-2). Interestingly, except for KLE cells, hinokitiol induced autophagy by promoting the accumulation of the microtubule-associated protein light chain 3B (LC3B) and reducing the sequestosome-1 (p62/SQSTM1) protein level. Furthermore, hinokitiol triggered ROS production and upregulated the phosphorylation of extracellular-signal-regulated kinase (p-ERK1/2) in EC cells. These results demonstrate that hinokitiol has potential anti-proliferative and pro-apoptotic benefits in the treatment of endometrial cancer cell lines (Ishikawa, HEC-1A, and KLE).

Relevant essential oil components: a minireview on increasing applications and potential toxicity.

Phenolic compounds carvacrol, thymol, eugenol, and vanillin are four of the most thoroughly investigated essential oil components given their relevant biological properties. These compounds are generally considered safe for consumption and have been used in a wide range of food and non-food applications. Significant biological properties, including antimicrobial, antioxidant, analgesic, anti-inflammatory, anti-mutagenic, or anti-carcinogenic activity, have been described for these components. They are versatile molecules with wide-ranging potential applications whose use may substantially increase in forthcoming years. However, some in vitro and in vivo studies, and several case reports, have indicated that carvacrol, thymol, and eugenol may have potential toxicological effects. Oxidative stress has been described as the main mechanism underlying their cytotoxic behavior, and mutagenic and genotoxic effects have been occasionally observed. In vivo studies show adverse effects after acute and prolonged carvacrol and thymol exposure in mice, rats, and rabbits, and eugenol has caused pulmonary and renal damage in exposed frogs. In humans, exposure to these three compounds may cause different adverse reactions, including skin irritation, inflammation, ulcer formation, dermatitis, or slow healing. Toxicological vanillin effects have been less reported, although reduced cell viability after exposure to high concentrations has been described. In this context, the possible risks deriving from increased exposure to these components for human health and the environment should be thoroughly revised.

Chemical Composition and Biological Activities of Essential Oils from Peels of Three Citrus Species.

BACKGROUND: Fruit peels are generally underutilized byproducts of the food industry, although they are valuable sources of bioactive compounds. The aim of this study is to evaluate a new application for three Citrus peel EOs as bio-herbicides. METHODS: After a micro-morphological evaluation of Citrus peels by SEM analysis, the phytochemical composition of the EOs of Citrus × bergamia Risso & Poit., Citrus × myrtifolia Raf., and Citrus limon (L.) Osbeck was characterized by GC/FID and GC/MS analyses. The in vitro phytotoxicity against germination and initial radical elongation of several crop and weed species was evaluated. Furthermore, the eco-compatibility of these EOs has been assessed by the brine shrimp (Artemia salina) lethality assay. RESULTS: SEM analysis highlighted the morphometric differences of the schizolysigenous pockets among the peels of the three Citrus species. Oxygenated monoterpenes are the main constituents in C. × bergamia (51.09%), whereas monoterpene hydrocarbons represent the most abundant compounds in C. × myrtifolia (82.15%) and C. limon (80.33%) EOs. They showed marked and selective phytotoxic activity in vitro, often at very low concentration (0.1 µg/mL) against all plant species investigated, without showing any toxicity on Artemia salina, opening the perspective of their use as safe bio-herbicides.

A lavender ABC transporter confers resistance to monoterpene toxicity in yeast.

MAIN CONCLUSION: Functional expression of a multidrug resistance-type ABC transporter from Lavandulaangustifolia improved yeast resistance to geraniol, a monoterpene constituent of lavender essential oil. Plant ATP-binding cassette (ABC) transporters are a large family of membrane proteins involved in active and selective transport of structurally diverse compounds. In this study, we functionally evaluated LaABCB1, a multidrug resistance (MDR)-type ABC transporter strongly expressed in the secretory cells of lavender glandular trichomes, where monoterpene essential oil constituents are synthesized and secreted. We used LaABCB1 to complement a yeast knockout mutant in which 16 ABC transporters were deleted. Expression of LaABCB1 enhanced tolerance of yeast mutants to geraniol, a key constituent of essential oils in lavenders and numerous other plants. Our findings suggest a role for the MDR-type ABC transporters in the toxicity tolerance of at least certain essential oil constituents in lavender oil glands.

Linalool- and α-terpineol-induced programmed cell death in Chlamydomonas reinhardtii.

Plant allelochemicals effectively inhibit and/ or control algal growth, and have potential to use as algaecide. To uncover the lethal mechanism of 2 anti-algal compounds linalool and α-terpineol identified from Cinnamomum camphora extracts, and promote their development as algaecide, the H2O2 production, photosynthetic abilities, caspase-like activities, nuclear changes and DNA degradation were investigated in Chlamydomonas reinhardtii treated with the 2 compounds. H2O2 content burst in linalool treatment at 0.5 h and in α-terpineol treatment at 1 h, with increases of 2.7 folds and 1.3 folds, respectively, compared to that at 0 h. The photosynthetic pigments gradually degraded, and Fv/Fm gradually declined to zero, indicating that the cell death was not a necrosis due to the gradual disappearance of physiological process. In C. reinhardtii cells, the caspase-9-like and caspase-3-like were activated in the treatments with the 2 compounds for 1 h. With prolonging the treatment time, the fluorescent intensity of the cell nucleuses stained by DAPI gradually enhanced and then faded, and the genomic DNA isolated from the cells gradually degraded. These hallmarks indicated that the death of C. reinhardtii cells in linalool and α-terpineol treatments was a programmed cell death (PCD) triggered by the increased reactive oxygen species (ROS). Compared to α-terpineol treatment, linalool treatment showed stronger promoting effects on PCD at the same time point, which may be caused by the higher ROS content inducing higher caspase-9-like and caspase-3-like activities in a short time.

The insecticidal activity of Satureja hortensis essential oil and its active ingredient -carvacrol against Acrobasis advenella (Zinck.) (Lepidoptera, Pyralidae).

Nowadays, Acrobasis advenella is considered a pest of the highest economic significance in black chokeberry plantations, negatively affecting the quantity and quality of fruits. The aim of this study was to investigate the effect of Satureja hortensis essential oil and its main constituent, carvacrol, on the life cycle and physiology of A. advenella. The metabolic activity of both preparations was evaluated against insect α- and ß- glucosidase, catalase, peroxidase and polyphenol oxidase. The results showed S. hortensis essential oil and carvacrol, are characterized by insecticidal activity against A. advenella larvae. It found an increase in catalase activity in A. advenella homogenates under the influence of carvacrol and an induction of polyphenol oxidase by S. hortensis EO with no changes in POX activity. Also, it was shown that the activity of α- and ß-glucosidase significantly increased in larvae fed on inflorescences treated with the essential oil and carvacrol. These preparations particularly strongly affected ß-glucosidase activity in the insect homogenates. S. hortensis essential oil reduced emergence the longevity of moths. The obtained results suggest that S. hortensis essential oil and carvacrol can be useful in protecting organic crops of black chokeberry but essential oils may be more effective biopesticides than their active ingredients separately.

Insecticide activity of Curcuma longa (leaves) essential oil and its major compound α-phellandrene against Lucilia cuprina larvae (Diptera: Calliphoridae): Histological and ultrastructural biomarkers assessment.

Lucilia cuprina, known as the Australian blowfly, is of high medico-sanitary and veterinary importance due to its ability to induce myiasis. Synthetic products are the most frequent form of fly control, but their indiscriminate use has selected for resistant populations and accounted for high levels of residues in animal products. This study aimed to assess the effect of essential oil from leaves of Curcuma longa (CLLEO), and its major compound α-phellandrene against L. cuprina L3. An additional goal was to determine the morphological alterations in target organs/tissues through ultrastructural assessment (SEM) and light microscopy, as well as macroscopic damage to cuticle induced by CLLEO. Groups of 20 L3 were placed on filter paper impregnated with increasing concentrations of CLLEO (0.15 to 2.86 µL/cm2) and α-phellandrene (0.29 to 1.47 µL/cm2). Efficacy was determined by quantifying L3 mortality 6, 24 and 48 h after contact with CLLEO and by measuring the structural damage to L3. CLLEO and α-phellandrene inhibited adult emergence by 96.22 and 100%, respectively. Macroscopic cuticle damage, appeared as diffuse pigment and darkening of larval body, was caused by both extracts. The SEM revealed dryness on the cuticle surface, distortion of the sensorial structures and general degeneration in treated L3. Furthermore, alterations in target organs (digestive tract, fat body and brain) were noticed and shall be used as biomarkers in future attempts to elucidate the mechanism of action of these compounds. The vacuolar degeneration and pyknotic profiles observed in the brain tissue of treated larvae with both extracts and the decreased motility within <6 h after treatment leads us to suggest a neurotoxic activity of the products. This work demonstrates the potential use of CLLEO and α-phellandrene as bioinsecticides to be used against L. cuprina, representing an ecofriendly alternative for myiasis control in humans and animals.

Development, characterisation and efficacy evaluation of biochemical fungicidal formulations for postharvest control of anthracnose (Colletotrichum gloeosporioides Penz) disease in mango.

The objectives of the study are to develop and characterise formulations with volatile molecules in an emulsifiable concentrate form, for their antimicrobial properties and to evaluate their efficacies against Colletotrichum gloeosporioides Penz., to control anthracnose in mangoes after harvest. Results showed EC39 and EC40 among formulations were characterised for their excellent emulsification properties, the droplet size of 192.34 ± 0.48 nm and 227.4 ± 0.71 nm and Zeta potential of -52.5 ± 2.76 mv and -48.84 ± 2.62 mv, respectively, with better storage stability at 10 ± 20 °C and RH 80 ± 5%. In vitro assay, 100% inhibition of visual spore germination by 0.15% and 0.2% MIC value for EC39 and EC40, respectively Studies on the efficacy of their fungicide properties also indicated the IC50 value of 0.161% and 0.162% for EC39 and EC40 respectively for mycelial growth inhibition. In vivo testing too, EC39 and EC40 effectively controlled anthracnose incidence in mango in a dosage-dependent manner.

Hepatotoxicity of monoterpenes and sesquiterpenes.

Public interest in natural therapies has increased significantly over past decades. Herbs and herbal products are extensively consumed worldwide and they are generally considered as safe. However, this may not always be true as many cases of herb-induced liver injury are reported every year. The liver is a frequent target tissue of toxicity from all classes of toxicants as liver structure and function predispose it to high sensitivity to xenobiotics. The present review is focused on the hepatotoxic properties of monoterpenes and sesquiterpenes, plant secondary metabolites that represent the major components of essential oils wildly used in folk medicines, pharmaceutical industry and cosmetics. Most of these terpenes easily enter the human body by oral absorption, penetration through the skin, or inhalation leading to measurable blood concentrations. Several studies showed that some monoterpenes (e.g., pulegone, menthofuran, camphor, and limonene) and sesquiterpenes (e.g., zederone, germacrone) exhibited liver toxicity, which is mainly based on reactive metabolites formation, increased concentration of reactive oxygen species and impaired antioxidant defense. There is a high probability that many other terpenes, without sufficiently known metabolism and effects in human liver, could also exert hepatotoxicity. Especially terpenes, that are important components of essential oils with proved hepatotoxicity, should deserve more attention. Intensive research in terpenes metabolism and toxicity represent the only way to reduce the risk of liver injury induced by essential oils and other terpenes-containing products.

Toxicity and repellency of essential oil from Evodia lenticellata Huang fruits and its major monoterpenes against three stored-product insects.

In this work, the essential oil (EO) was extracted from the fruits of Evodia lenticellata, and the fumigant toxicity, contact toxicity and repellency against three stored-product insect species were evaluated for the obtained EO and several of its chemical components. The target insects were the adults of Tribolium castaneum (Coleoptera: Tenebrionidae), Lasioderma serricorne (Coleoptera: Anobiidae) and Liposcelis bostrychophila (Psocoptera: Liposcelididae). The EO was obtained with hydrodistillation and its chemical components were analyzed with the gas chromatography-mass spectrometry (GC-MS). Twenty-seven compounds, accounting for 83.1% of the total amount of the oil, were identified from the EO sample. The main compounds included linalool (12.0%), ß-pinene (11.5%), 3-carene (9.6%), caryophyllene oxide (8.7%) and ß-caryophyllene (7.9%). Among them, the amounts of monoterpenes and sesquiterpenes were as high as 52.7% and 22.7% to the total amount of EO respectively. The results of bioactivity test showed that the EO and its testing compounds had interspecific toxicity and repellent activity. So that, it might be expected that the EO extracted from the fruits of E. lenticellata could be developed to a new type of eco-friendly natural insecticide or repellent for the control of stored-product insects.

In vitro effects of two major phenolic compounds from the family Lamiaceae plants on the human gastric carcinoma cells.

Phenolic compounds of essential oils from the family Lamiaceae are commonly used substances in the food industry because of their flavouring, antimicrobial and antioxidant properties. In this context, it has become important to have healthy and safe products for consumers who are exposed to these phenolic compounds. The present study was aimed to investigate the toxic effects of carvacrol, thymol and their mixture on human gastric carcinoma (AGS) cells. Cells were analyzed after 24 h of exposure to different concentrations of carvacrol, thymol and their mixture by the ATP cell viability, 2',7' dichlorodihydrofluorescein diacetate (H2DCF-DA), reducte glutatione/oxide glutathione ((GSH)/GSSG-Glo) and comet assays. Apoptosis induction was studied by acridine orange/ethidium bromide staining and western blotting. Carvacrol, thymol and their mixture induced cytotoxicity, genotoxicity, apoptosis, increased reactive oxygen species (ROS) and decreased GSH levels after 24 h of their exposure in a dose-dependent manner. A close negative relationship was found between cell viability and ROS generation. We examined dose-dependent cytotoxic effects of carvacrol, thymol and their mixture in human AGS cells. Increased intracellular ROS causes oxidative stress in cells. The results indicated that these compounds should be used carefully in the food industry.

Inter-Individual Variability in Acute Toxicity of R-Pulegone and R-Menthofuran in Human Liver Slices and Their Influence on miRNA Expression Changes in Comparison to Acetaminophen.

Monoterpenes R-pulegone (PUL) and R-menthofuran (MF), abundant in the Lamiaceae family, are frequently used in herb and food products. Although their hepatotoxicity was shown in rodent species, information about their effects in human liver has been limited. The aim of our study was to test the effects of PUL, MF and acetaminophen (APAP, as a reference compound) on cell viability and microRNA (miRNA) expression in human precision-cut liver slices. Slices from five patients were used to follow up on the inter-individual variability. PUL was toxic in all liver samples (the half-maximal effective concentration was 4.0 µg/mg of tissue), while MF and surprisingly APAP only in two and three liver samples, respectively. PUL also changed miRNA expression more significantly than MF and APAP. The most pronounced effect was a marked decrease of miR-155-5p expression caused by PUL even in non-toxic concentrations in all five liver samples. Our results showed that PUL is much more toxic than MF and APAP in human liver and that miR-155-5p could be a good marker of PUL early hepatotoxicity. Marked inter-individual variabilities in all our results demonstrate the high probability of significant differences in the hepatotoxicity of tested compounds among people.

Effect of citral on mouse hepatic cytochrome P450 enzymes.

CONTEXT: Citral is used as a potential natural treatment for various infectious diseases. OBJECTIVE: To examine the effect of citral on the mRNA expression and activities of cytochrome P450 (CYP450) enzymes and establish the relationship between citral-induced liver injury and oxidative stress. MATERIALS AND METHODS: ICR mice were randomly divided into citral (20, 200, and 2000 mg/kglow), Tween-80, and control groups (0.9% saline), 10 mice in each group. The citral-treated groups were intragastrically administered citral for 3 d, control groups treated with 0.5% Tween-80 and 0.9% saline in the same way. Liver injury and CYP450 enzymes were analyzed by analyzing the histopathological changes and the changes of related enzymes. RESULTS: Citral treatment (2000 mg/kg) for 3 d increased serum glutamic pyruvic transaminase and glutamic oxaloacetic transaminase levels, as well as glutathione, gydroxyl radicals, malonaldehyde and total superoxide dismutase contents, but decreased the content of total antioxidant capacity. In doses of 20 and 200 mg/kg groups mice, the contents of NO were decreased significantly and other changes were similar to the 2000 mg/kg group mice, but the liver damage was most severe in the 2000 mg/kg group. Citral induced the mRNA expression and activities of CYP450 1A2, 2D22, and 2E1 in the liver of mice at doses of 20 and 200 mg/kg. There were no changes in testing indexes in Tween-80 treated group mice. Due to its toxic effects, the CYP induction effect of citral negatively correlated with its dose. Although the mRNA expression of CYP450 3A11 was induced by citral, its activity was not affected by low and moderate doses of citral. CYP450 3A11 activity was significantly decreased by high-dose citral. CONCLUSIONS: Citral is hepatotoxic and induced oxidative stress in higher dose, which has a negative effect on CYP450 enzymes. These data suggest caution needs to be taken in order to avoid citral-drug interactions in human beings.

Chemical and thermal sensitivity of medaka TRPA1 analyzed in heterologous expression system.

TRPA1 of insects and several tetrapod vertebrates except for those of rodents have been reported to be activated by noxious chemicals and also by high temperature with a relatively clear threshold. We previously analyzed the characteristics of two TRPA1 paralogs of zebrafish (zTRPA1a, b) and demonstrated that zTRPA1a is specialized for chemical sensing while zTRPA1b responds to thermal stimulations, that zTRPA1b responds to both cold and heat stimuli, and that heat stimulation gradually activates zTRPA1b without a clear threshold. In the medaka genome, a single TRPA1 (olTRPA1) gene is present. To examine if functional properties of olTRPA1 are similar to TRPA1 of land animals or either of zTRPA1a or zTRPA1b, we isolated a TRPA1 cDNA from medaka and performed functional analyses. OlTRPA1 showed a sensitivity to four noxious chemicals (allyl isothiocyanate, caffeine, carvacrol, methyl anthranilate). We observed that cold stimulation does not activate olTRPA1, but heat stimulation gradually activates olTRPA1 with an unclear threshold. Results suggested that a single TRPA1 functions as a chemical and thermal sensor in medaka, and that a gradual heat-activation without clear threshold might be a common feature for TRPA1 of fish living in water.

Toxicity of Monoterpene Structure, Diversity and Concentration to Mountain Pine Beetles, Dendroctonus ponderosae: Beetle Traits Matter More.

A high diversity of plant defenses may be a response to herbivore diversity or may be collectively more toxic than single compounds, either of which may be important for understanding insect-plant associations. Monoterpenes in conifers are particularly diverse. We tested the fumigant toxicity of four monoterpenes, alone and in combination, to mountain pine beetles, Dendroctonus ponderosae, in the context of the beetles' individual body traits. Chemical structures of tested monoterpene hydrocarbons had modest effects on beetle survival, mass loss, water content and fat content, with (R)-(+)-limonene tending to be more toxic than (-)-α-pinene, (-)-ß-pinene, and (+)-3-carene. Monoterpene diversity (all qualitative combinations of one to four monoterpenes) did not affect toxicity. Concentration (0 to 1200 ppm) of individual monoterpenes was a strong determinant of toxicity. Beetle body size and body condition index strongly and positively affected survival during monoterpene treatments. Larger beetles in better condition lost proportionally less mass during exposure, where proportion mass loss negatively affected survivorship. Toxicity was much more associated with water loss than with fat loss, suggesting that a main cost of detoxification is excretion, a process that has received little attention. These results provide insight into the determinants of beetle success in historic and novel hosts that differ in monoterpene composition and concentration. We also suggest that water availability will affect beetle success directly through their ability to tolerate detoxification as well as indirectly through host responses to drought.

Evaluation of larvicidal activity and ecotoxicity of linalool, methyl cinnamate and methyl cinnamate/linalool in combination against Aedes aegypti.

The frequent use of synthetic pesticides to control Aedes aegypti population can lead to environmental and/or human contamination and the emergence of resistant insects. Linalool and methyl cinnamate are presented as an alternative to the synthetic pesticides, since they can exhibit larvicidal, repellent and/or insecticidal activity and are considered safe for use. The aim of this study was to evaluate the larvicidal activity of methyl cinnamate, linalool and methyl cinnamate/linalool in combination (MC-L) (1:4 ratio, respectively) against Aedes aegypti. The in vitro preliminary toxicity through brine shrimp lethality assay and hemolytic activity, and the phytotoxic potential were also investigated to assess the safety of their use as larvicide. Methyl cinnamate showed significant larvicidal activity when compared to linalool (LC50 values of 35.4µg/mL and 275.2µg/mL, respectively) and to MC-L (LC50 138.0µg/mL). Larvae morphological changes subjected to the specified treatments were observed, as the flooding of tracheal system and midgut damage, hindering the larval development and survival. Preliminary in vitro toxicity through brine shrimp showed the high bioactivity of the substances (methyl cinnamate LC50 35.5µg/mL; linalool LC50 96.1µg/mL) and the mixture (MC-L LC50 57.7µg/mL). The results showed that, despite the higher larvicidal activity of methyl cinnamate, the use of MC-L as a larvicide seems to be more appropriate due to its significant larvicidal activity and low toxicity.

Application of bacterial reverse mutation assay for detection of non-genotoxic carcinogens.

Non-genotoxic carcinogens may play a significant role in development of cancer. Currently short-term assays for mutagenicity classify genotoxic carcinogens and lack the abilities to detect epigenetic carcinogens. The need to develop an endpoint always remains to recognize potentially carcinogenic agents employing rapid and practical bioassays. For this, the present study utilized TA98 and TA1537 tester strains of Salmonella typhimurium to evaluate four non-genotoxic carcinogenic agents (Coumarin, ß-Myrcene, Bis(2-ethylhexyl) phthalate and trans-anethole). These chemicals were tested individually and in combination with promutagens 2-aminoanthracene (2AA) and benzo(a)pyrene (BP) in presence of metabolic activation system (S9) by plate incorporation method. Exposure to all four test chemicals revealed marked increase of revertant colonies in promutagen combined groups as compared to promutagens alone. However significantly greater fold responses were observed with 2AA combination groups (Coumarin +2AA, ß-Myrcene +2AA, Bis(2-ethylhexyl) phthalate +2AA and trans-anethole +2AA) with TA98 strain as compared with TA1537, which seems to have enhanced the mutagenic response of 2AA in metabolically activated conditions. It is concluded that out of both tester strains TA98 strain of Salmonella typhimurium has the potential to detect non-genotoxic carcinogens when combined with potent promutgens either by inhibiting or modulating activities of liver microsomal enzymes biochemically which may indirectly contribute to neoplastic alterations. Further this simple, short-term alternative assay may provide rapid information during extrapolative toxicology for differentiating genotoxic and non-genotoxic carcinogens.