Pleiotropic activities with beneficial effects have been attributed to borage seed oil for the treatment of several disorders such as acute respiratory distress syndrome, rheumatoid arthritis, diabetic neuropathy, menopause and atopic dermatitis 
. Here, we present the first study showing results regarding the toxicity, genotoxicity, antigenotoxicity and longevity properties, of borage seed oil along with one of its active components (GLA). The study was performed in vivo
, in the eukaryotic system D. melanogaster
because more than half of its genes are known to have mammalian homologues 
. D. melanogaster
provides an excellent in vivo
genetic model system for toxicity studies 
has been used to model human conditions such as neurodegenerative and infectious diseases 
, immune system 
cardiac function 
, and aging 
. From a toxicological point of view Drosophila
and higher mammals have similar dose–response relationship with many compounds such as monofunctional alkylating agents 
. Thus, we believe that laboratory-based experimental evidences using this model is useful in generating information that could be of value for their efficient extrapolation to higher organisms.Therefore the information obtained regarding genotoxicity in an excellent indicator of the genetic safety of treatments with borage seed oil. Borage seed oil was toxic only at the highest assayed concentration (12,5%). This concentration equals to 7.5 g/day in humans subjects of 60 kg body weight. This fat intake represents 12,5% of the daily fat intake, which is under the Acceptable Macronutrient Distribution Range for fat (20–35%) of energy for total fat 
.This concentration is slightly higher than the usually recommended dosage (1–6 g/day) in chronic treatments for various degenerative pathologies 
. Thus, survival assays were performed with concentrations below 125 µl/ml.
More than 200 effects of hydrogen peroxide as a genotoxin have been previously described 
, and there are several antigenotoxic studies that demonstrate its genotoxic potency, being both mutagenic (oxidative genetic damage induced by the production hydroxyl radicals through the metal-catalyzed Fenton reaction) and recombinogenic (genotoxic mechanism involved as an early step in the origin of some types of human cancers) 
. Borage seed oil and GLA genotoxicity assays confirm the safety of these compounds which is the first step in characterizing their usefulness as a neutraceuticals. Besides this, our antigenotoxicity assays demonstrated for the first time that both borage seed oil and GLA exert a role in the genomic stability of D. melanogaster
, acting as a desmutagenic agents against hydrogen peroxide by scavenging the reactive oxygen species originated by the model genotoxicant used. The similarity in the antigenotoxic inhibitory potencies of GLA (38.1%) and borage seed oil (38.2%) as well as the high content of GLA in borage seed oil (21.1%) indicates that GLA may account for the antigenotoxic ability of borage seed oil.
Additionally, our results show that both substances tested have a cytotoxic effect on HL-60 tumour cells. Specific mechanisms inducing cytotoxicity have been observed for GLA in other studies: i) GLA induces apoptosis in this cell line checked by DNA fragmentation hallmark 
; ii) GLA enhances the cytotoxicity of docetaxel in human breast cancer cells by mechanisms other than lipoperoxidation, and the GLA-induced transcriptional repression of HER-2/neu oncogene might be one component of the mechanisms 
; iii) human chronic myelogenous leukaemia K562 cells are also switched to the apoptotic way by activation of caspase-3 and release of cytochrome c 
; iv) GLA treatment in hepatocellular carcinoma Hhh7 cell upregulated genes encoding antioxidant proteins 
; and v) in the Lobund-Wistar animal model of prostate cancer by treating rats with NMU and supplementing with GLA a decrease of prostate growth was observed 
. Additionally, it is known that intra-tumoural administration of GLA can induce regression of human gliomas 
and GLA linked to lithium salt is able to inhibit cell growth of two pancreatic tumour cell lines (MIA PaCa2 and Panc 1) 
. Therefore, our results are consistent with those of other authors, although they used different cell lines, and confirm the ability of cancer cell growth inhibition by GLA.
The cytotoxic effect of polyunsaturated fatty acids particularly arachidonic acid and the eicosanoids generated from it, is long known, being characterized as apoptotic and associated with oxidative stress. Chen et al., (1998) show that pretreatment with 0.03 mM arachidonic acid causes significant toxicity to the human hepatoma HepG2 cells (E9 cells) that over express human CYP2E1. The PUFA toxicity is associated with increased lipid peroxidation and can be diminished by antioxidants that prevent lipid peroxidation (such as ascorbic acid, Trolox and alpha tocopherol phosphate. Compared with arachidonic acid, oleic acid (C18:1) showed no significant toxicity to the E9 cells even at concentrations (0.05 mM) at which arachidonic acid is highly cytotoxic 
Similarly, Pompeia et al., (2002) demonstrated the cytotoxic effect of arachidonic acid in leukocytes (HL-60, Jurkat and Raji cells). Use this fatty acid was also demonstrated to have an apoptotic effect at low concentrations (10–400 µM) and a necrotic effect at high concentrations (400–1600 µM) 
Anti-ageing and anti-degenerative assays can be carried out using different D. melanogaster
strains in order to perform life span trials with specific chronic diets in controlled environments 
, as the key interest for humans is to increase life span. In this work, we describe for the first time a study designed to examine the effects of both borage seed oil and GLA dietary supplementation on the life span of D. melanogaster
. This animal model is an excellent system to investigate longevity-promoting properties of compounds and nutraceutical extracts. Drosophila
has a short life span, can be cultured with simple diets, and represents a rich genetic resource with a fully sequenced genome. The life span of this insect is relatively short and adults seem to show many of the cell senescence features seen in mammals 
. In this study, we demonstrate that low doses of borage seed oil are able to increase the health span portion ≥ 75% of the life span curves in D. melanogaster
(). No positive effects on life/health span in Drosophila melanogaster have been found at any concentration of GLA () The Drosophila
strains used in this work is not extended-life mutants. Average life span data of D. melanogaster
vary widely and are strongly dependent on rearing conditions. The average control life span (between 33 and 80 days) found in previous works 
, are lower than that found in the present study. Dietary supplementation does not always result in an increased life span. Dietary supplementation using different concentrations of substances such as nectarine, cocoa, broccoli or lamotrigine also resulted in values lower than with a normal diet (10% sugar and 10% yeast extract), dietary restriction (2.5% sugar and 2.5% yeast extract), or anoxia treatments 
. Our results show that borage seed oil did not reduce the life span, but increased significantly the health span portion of the life span curve.
The negative effects of GLA on the longevity trials of D. melanogaster
are in agreement with the toxicity results for this fatty acid () where GLA is shown to be more toxic than borage seed oil. Nevertheless, the genotoxic/antigenotoxic and cytotoxic assays with borage seed oil and GLA showed the same positive effect. This apparent mismatch could be due to the fact that GLA as free fatty acids is highly prone to the auto-oxidation process producing hydroperoxides and other oxygenated compounds that can reduce the average life span 
. Besides this, GLA and other fatty acids contained in borage seed oil are part of a triacylglycerol molecules that is more stable and less susceptible to auto-oxidation 
. Tso et al. (2002) studied the intestinal absorption and lymphatic transport of two different seed oils containing GLA using a lymph fistula rat model, and demonstrated that the oleic, linoleic and gamma linoleic acid content of the dietary oils consumed are preserved in the fatty acid composition of lymph triglycerides 
Taking into account the whole toxicity, geno/antigenotoxicity, lifespan and cytotoxicity assays performed in the present study, the beneficial effects of borage seed oil described may be due not only to the presence of GLA but also to other major constituents, like oleic and linoleic acids, delta tocopherol and gamma-tocopherol () with antioxidant and contrasted beneficial properties for various degenerative pathologies 
Based on the findings of the present study, we conclude that i) borage seed oil is non- toxic to D. melanogaster at concentrations below 125 µl/ml and the studies on GLA indicated non-toxic at the lowest concentration used ii) borage seed oil and GLA are DNA safe (non-genotoxic) and antimutagenic compared to hydrogen peroxide, thereby confirming their antioxidant capacity; iii) borage seed oil and GLA exhibited cytotoxic activity in low doses (IC50 of 1 µl/ml and 0.087 mM, respectively) iv) Low doses of borage seed oil (0.19%) increased the health span of D. melanogaster; and v) GLA significantly decreased the life span of D. melanogaster.
Based on the antimutagenic and cytotoxic effects along with the ability to increase the health span, we propose the use of borage seed oil, rather than GLA, as a substance with antimutagenic/anticarcinogenic properties because it protects DNA by modulating the in vivo oxidative genetic damage in D. melanogaster, increases the health span and exerts in vitro cytotoxic activity towards promyelocytic HL60 cells.