Subchronic Oral Toxicity Study of Genetically Modified Rice Rich in ß-Carotene in Wistar Rats.

(1) Background: a hybrid black rice rich in ß-carotene carrying the psy and crtI genes (HJM) was evaluated in Wistar rats by a 90-day feeding study, aiming to assess its dietary safety. (2) Methods: the HJM rice and its parental line HS were included in rats' diets at levels of 73.5% and 75.5%, respectively. The AIN-93 diet was administered as a nutritional control. No adverse effects on animal behavior or weight gain were observed during the study. Blood samples were collected and analyzed, and standard hematological and biochemical parameters were compared. (3) Results: Some parameters were found to be significantly different, though they remained within the normal range for rats of this breed and age. In addition, upon sacrifice, various organs were weighed, and macroscopic and histopathological examinations were performed, with only minor changes to report. (4) Conclusions: HJM rice exhibited no adverse or toxic effects in Wistar rats in this 90-day study.

ß-Carotene oxidation products - Function and safety.

ß-Carotene oxidation products have newly discovered bioactivity in plants and animals. Synthetic fully oxidized ß-carotene (OxBC) has application in supporting livestock health, with potential human applications. The safety of synthetic OxBC has been evaluated. An Ames test showed weak-to-moderate mutagenicity in only one cell line at high concentrations. A mouse micronucleus assay established a non-toxic dose of 1800 mg/kg body weight, and no bone marrow micronuclei were induced. Plant sources of ß-carotene inevitably contain varying levels of natural OxBC. Vegetable powders and dried forages can be especially rich. Intakes of natural OxBC for humans and livestock alike have been estimated. The exposure range for humans (1-22 mg/serving) is comparable to the safe intake of ß-carotene (<15 mg/d). In livestock, OxBC in alfalfa can contribute ~550-850 mg/head/d for dairy cattle but in forage-deficient poultry feed much less (~1 ppm). Livestock intake of supplemental synthetic OxBC is comparable to OxBC potentially available from traditional plant sources. Human intake of synthetic OxBC in meat from livestock fed OxBC is similar to a single serving of food made with carrot powder. It is concluded that consumption of synthetic OxBC at levels comparable to natural OxBC is safe for humans and animals.

Enhancement of the antioxidant activity and stability of ß-carotene using amphiphilic chitosan/nucleic acid polyplexes.

ß-carotene is a natural compound with significant antioxidant activity. However, its poor solubility in water and low stability reduce its potential application. Innovative polyplexes based on the combination of amphiphilic chitosan assembled with DNA have been developed using a solvent-free, simple and low-cost method with the aim to load, retain and enhance the antioxidant capability of ß-carotene. The polyplexes, with dimension about 100 nm, and excellent stability, were able to hold up to 400 µg of ß-carotene per mg of the carrier, with minimal loss till two weeks. The antioxidant activity was significantly enhanced after loading, as demonstrated using two well known methods. Cytotoxicity assay confirmed the not toxicity of the system. The results suggest the polyplexes as an excellent candidate to develop formulation able to preserve and enhance the peculiarities of compounds which are used mainly in food, cosmetic and pharmaceutic but with still some limitations.

In Vitro Bioavailability, Cellular Antioxidant Activity, and Cytotoxicity of ß-Carotene-Loaded Emulsions Stabilized by Catechin-Egg White Protein Conjugates.

Previously, it was shown that catechin-egg white protein (CT-EWP) conjugates were effective antioxidant emulsifiers that could form and stabilize emulsions, and also inhibit the degradation of encapsulated carotenoids. The objective of the current study was to evaluate the impact of conjugation on the in vitro bioavailability, cellular antioxidant activity, and cytotoxicity of ß-carotene-loaded emulsions. Lipid droplets coated with EWP or with CT-EWP conjugates exhibited quite similar behavior when they were passed through a simulated gastrointestinal tract. The ß-carotene encapsulated in emulsions stabilized by CT-EWP conjugates exhibited a higher overall in vitro bioavailability, which was attributed to a greater stability of the carotenoids to chemical transformation. The emulsions stabilized by CT-EWP conjugates also exhibited greater ability in inhibiting oxidation in a cell-based assay (dichlorofluorescein diacetate). Cytotoxicity analysis suggested that ß-carotene emulsions stabilized by CT-EWP conjugates only exhibited a slight cytotoxicity when used at high concentrations. These results suggest that CT-EWP conjugates can be used to formulate emulsion-based delivery systems for chemically labile hydrophobic bioactives with enhanced antioxidant activity and bioavailability.

Safety Assessment of Oil from Pequi (Caryocar brasiliense Camb.): Evaluation of the Potential Genotoxic and Clastogenic Effects.

Genotoxic data of medicinal plants and functional foods are required as part of the risk assessment by international regulatory agencies. Due to its food consumption and ethnopharmacological relevance, pequi oil (Caryocar brasiliense Camb.) is one of these compounds to be studied. The aim of this study was to evaluate the cytotoxic, genotoxic, and clastogenic effects of the oil from the pulp of C. brasiliense (OPCB) in vivo and in vitro. Initially, the Artemia salina in vitro assay was conducted to determine the cells viability rate of different doses of the OPCB. Subsequently, comet assay (Organization for Economic Cooperation and Development, OECD 489) and micronucleus test (OECD 474) were performed in blood and bone marrow of Wistar rats treated orally with a 125, 250, 500, or 1000 mg/kg/bw of the OPCB for 4 weeks. The chemical analysis indicated the presence of ß-carotene and lycopene in the oil. In the A. salina test, all OPCB doses maintained cell viability rates statistically similar to the negative control. The in vivo tests performed showed that OPCB did not show significant genotoxic or clastogenic effects in cells analyzed with the four doses tested. Altogether, these results indicate that, under our experimental conditions, C. brasiliense fruit oil did not reveal genetic toxicity in rat cells.

The safety of ß-carotene from Yarrowia lipolytica.

Crystalline ß-carotene from genetically modified Yarrowia lipolytica is an alternative source of ß-carotene for use as a nutritional supplement. To support the use of ß-carotene from Y. lipolytica as a food ingredient, the genotoxic and subchronic toxicity potential of this compound was determined. Genotoxicity was examined using Salmonella typhimurium and Escherichia coli (Ames test), a chromosomal aberration assay in Chinese Hamster Ovary WBL cells, and the micronucleus test in CD-1 mice. All three assays showed no significant results due to ß-carotene from Y. lipolytica. In a subchronic toxicity study in SD rats, ß-carotene from Y. lipolytica was administered by oral gavage for 13weeks at 0, 125, 250 or 500mg/kg per day. Adverse effects were not observed following clinical, clinical pathology and gross- and histopathological evaluations of dosed rats; thus, the no-observed-adverse effect level (NOAEL) for ß-carotene from Y. lipolytica was 500mg/kg, the highest dose used in the study. In conclusion, ß-carotene derived from Y. lipolytica was shown in genotoxicity models and a standard rat subchronic rat study to have a safety profile similar to that of the current commercial products (synthetic and natural) with no unexpected finding attributable to the alternative source.

Cytotoxicity of ß-carotene cleavage products and its prevention by antioxidants.

When we investigated the genotoxicity of beta-carotene cleavage products (CPs) in primary rat hepatocytes stimulated to proliferate, we observed dose-dependent increases of chromosomal aberrations, sister chromatid exchanges and micronuclei. In contrast to other genotoxic substances, however, this increased genotoxicity was not accompanied by increased cytotoxicity. As a consequence we observed metaphases showing massive chromosomal damage, indicating inhibition of apoptosis by CPs enabling these cells to proceed in the cell cycle. Since proliferative stimulation by growth factors may support this effect, the in vitro toxicological effects of CPs were studied on proliferatively quiescent primary rat hepatocytes. A significant increase of both apoptosis and necrosis was found. Supplementation with antioxidants did not significantly lower the level of apoptosis, while the level of necrosis was significantly reduced by Trolox and N-acetylcysteine at all concentrations tested as well as ascorbic acid (50 microM) and a combination of Trolox (50 microM) and ascorbic acid (50 microM). These observations indicate that a) the cytotoxic potential in combination with the genotoxic potential of CPs may promote the initiation of cells due to compensatory cell division in exposed tissues and may aggravate inflammatory processes under chronic exposure, and b) the applied antioxidants may protect from cytotoxicity primarily via the detoxification of aldehydic beta-carotene cleavage products.

In vitro and in vivo evaluation of mutagenicity of fucoxanthin (FX) and its metabolite fucoxanthinol (FXOH).

Mutagenicity of fucoxanthinol (FXOH), the major compound after oral ingestion of fucoxanthin (FX), was evaluated by in vitro Ames test, and of FX by in vivo micronucleus test. In in vitro Ames test, bacterial reverse mutation was examined by using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, and Escherichia coli WP2uvrA/pKM101, with or without metabolic activation by S9 mix in the preincubation method, and mutagenicity of FXOH was found to be negative in all cases. In in vivo micronucleus test, mice were orally administered with FX at doses of 500, 1,000 and 2,000 mg/kg, and the bone marrow cells were taken 24 hr after the administration to observe the incidence of micronucleus cells, and mutagenicity of FX was found to be negative at all doses. Based on the data of the present study it can be presumed that orally administered FX is a safe compound in terms of mutagenicity under the experimental conditions employed here.

Development of dietary phytochemical chemopreventive agents: biomarkers and choice of dose for early clinical trials.

In view of safety concerns surrounding the use of pharmaceuticals such as nonsteroidal anti-inflammatory drugs and tamoxifen as cancer chemopreventive agents, potentially innocuous phytochemicals derived from the diet are considered attractive alternatives. However, results from cancer chemoprevention trials of dietary agents have been disappointing to date, as promising activities observed in rodent models and cells in vitro have not translated into clinical success. This may be partly due to the development process for these agents, which is complex for a number of reasons; the definitive end point, inhibition of carcinogenesis, requires large numbers of individuals followed-up over many years. Furthermore, whereas biomarkers are frequently used as surrogate efficacy end points to expedite the process, biomarker assessment and validation has proven difficult because dietary agents exert multiple actions with an unknown hierarchy of biological importance. These factors have made determining the dose for clinical investigation extremely challenging, and at present, there are no defined strategies for rationally identifying the most appropriate doses. In this commentary, the complexities involved in the development of dietary chemoprevention agents are discussed, and a tentative route towards selection of the optimal clinical dose is proposed. The approach highlights the need to conduct long-term preclinical studies with realistic concentrations that are achievable in human tissues and the importance of efficacy biomarkers that are intrinsically linked to the key mechanisms of action. A more logical design of studies should increase the likelihood that the encouraging preclinical results observed for many phytochemicals translate into tangible clinical benefit.

Beta-carotene degradation products - formation, toxicity and prevention of toxicity.

Carotenoids are widely used as important micronutrients in food. Furthermore, carotenoid supplementation has been used in the treatment of diseases associated with oxidative stress such as various types of cancer, inflammatory diseases or cystic fibrosis. However, in some clinical studies harmful effects have been observed, e.g. a higher incidence of lung cancer in individuals exposed to extraordinary oxidative stress. The causal mechanisms of harmful effects are still unclear. Carotenoid breakdown products (CBPs) including highly reactive aldehydes and epoxides are formed during oxidative attacks in the course of antioxidative action. We investigated the formation of CBPs by stimulated neutrophils (and at further conditions), tested the hypothesis that CBPs may exert mitochondriotoxicity and tried to prevent toxicity in the presence of members of the antioxidative network. Stimulated neutrophils are able to degrade beta-carotene and to generate a number of CBPs. Concerning mitochondriotoxicity, we found that CBPs strongly inhibit state 3 respiration of rat liver mitochondria at concentrations between 0.5 and 20 microM. This was true for retinal, beta-ionone, and for mixtures of cleavage/breakdown products. The inhibition of mitochondrial respiration was accompanied by a reduction in protein sulfhydryl content, decreasing GSH levels and redox state, and elevated accumulation of malondialdehyde. Changes in mitochondrial membrane potential favor functional deterioration in the adenine nucleotide translocator as a sensitive target. The presence of additional antioxidants such as alpha-tocopherol, ascorbic acid, N-acetyl-cysteine or others could mitigate mitochondriotoxicity. The findings reflect a basic mechanism of increasing the risk of cancer induced by carotenoid degradation products.

Effects of quercetin on beta-apo-8'-carotenal-induced DNA damage and cytochrome P1A2 expression in A549 cells.

We compared the effects of beta-carotene with those of beta-apo-8'-carotenal (AC, an oxidative product of beta-carotene) on DNA damage and the expression of cytochrome P450 (CYP)1A2 in A549 cells exposed or not to benzo[a]pyrene (BaP), a cigarette-associated carcinogen. Furthermore, we investigated whether quercetin, a flavonoid, modulates these effects. A549 cells were first preincubated with various concentrations of beta-carotene or AC for 1h, followed by incubation with 20 microM BaP for 24h. Next, DNA strand breaks, measured by use of the comet assay, and the expression of CYP1A2, measured by use of western blotting, were assessed. Both beta-carotene and AC at 20 microM significantly enhanced DNA strand breaks and CYP1A2 expression induced by BaP. However, beta-carotene at 2 microM significantly suppressed BaP-induced DNA strand breaks. AC alone induced DNA strand breaks, lipid peroxidation, and the expression of CYP1A2 in A549 cells. The harmful effects of beta-carotene and AC on intracellular DNA were associated with the expression of CYP, because 1-aminobenzotriazole, a CYP inhibitor, partly suppressed these effects. Quercetin significantly inhibited the DNA strand breaks and the increase in CYP1A2 protein induced by AC or beta-carotene in combination with BaP or by AC alone. These findings indicate that the harmful effect of beta-carotene induced by BaP may be through the formation of oxidative products such as AC. Quercetin increased the safety of high doses of beta-carotene, possibly through interaction with beta-carotene's oxidative products or through inhibition of CYP1A2 expression.

Dual role of beta-carotene in combination with cigarette smoke aqueous extract on the formation of mutagenic lipid peroxidation products in lung membranes: dependence on pO2.

Results from some intervention trials indicated that supplemental beta-carotene enhanced lung cancer incidence and mortality in chronic smokers. The aim of this study was to verify the hypothesis that high concentrations of the carotenoid, under the pO2 present in lung (100-150 mmHg), may exert deleterious effects through a prooxidant mechanism. To test this hypothesis, we examined the interactions of beta-carotene and cigarette smoke condensate (tar) on the formation of lipid peroxidation products in rat lung microsomal membranes enriched in vitro with varying beta-carotene concentrations (from 1 to 10 nmol/mg prot) and then incubated with tar (6-25 microg/ml) under different pO2. As markers of lipid peroxidation, we evaluated the levels of conjugated dienes and malondialdehyde, possessing mutagenic and pro-carcinogenic activity. The exposure of microsomal membranes to tar induced a dose-dependent enhancement of lipid peroxidation, which progressively increased as a function of pO2. Under a low pO2 (15 mmHg), beta-carotene acted clearly as an antioxidant, inhibiting tar-induced lipid peroxidation. However, the carotenoid progressively lost its antioxidant efficiency by increasing pO2 (50-100 mmHg) and acted as a prooxidant at pO2 ranging from 100 to 760 mmHg in a dose-dependent manner. Consistent with this finding, the addition of alpha-tocopherol (25 microM) prevented the prooxidant effects of the carotenoid. beta-Carotene auto-oxidation, measured as formation of 5,6-epoxy-beta,beta-carotene, was faster at high than at low pO2 and the carotenoid was more rapidly consumed in the presence of tar. These data point out that the carotenoid may enhance cigarette smoke-induced oxidative stress and exert potential deleterious effects at the pO2 normally present in lung tissue.

[Systemic photoprotection through carotenoids]. / Systemische Photoprotektion durch Karotinoide.

Nutritional supplements are increasingly used to protect human skin against environmentally-induced damage, most importantly as a consequence of ultraviolet radiation exposure. beta-carotene is a major constituent of comercially available products administered for systemic photoprotection. Studies on the systemic use of beta-carotene provide evidence that 15-30 mg/d over a period of about 10-12 wk produces a protective effect against UV-induced erythema. Similar effects have been attributed to mixtures of carotenoids or after long-term intake of dietary products rich in carotenoids. Supplementation with carotenoids contributes to basal protection of the skin but is not sufficent to obtain complete protection against severe UV irradiation.

A subchronic toxicity study of dunaliella carotene in F344 rats.

Dunaliella carotene, extracted from dunaliella alga (Dunaliella bardawil or Dunaliella salina), for use as a food-coloring agent, has beta-carotene as its mainly constituent. As there have been no reports of toxicological evaluation, a 90-day subchronic toxicity study was here performed in F344 rats at dose levels of 0 (control), 0.63%, 1.25%, 2.5% and 5% in powdered basal diet. The average daily intakes of dunaliella carotene were 352, 696, 1420 and 2750 mg/kg/day, respectively, for males, and 370, 748, 1444 and 2879 mg/kg/day for females. No mortality or treatment-related clinical signs were observed throughout the experimental period in any of the groups. Body weight gain was slightly but significantly (p < 0.05) reduced from week 5 to the end of the experiment in 2.5% and 5% males. Increased PLT were observed in 1.25% and 5% males, and 2.5% and 5% females. Significant elevations or tendencies for increase in serum T. Cho and Ca were observed in all treated males and females, with clear dose-dependence in males. Organ weight measurement and histopathological observation revealed no toxicological changes. Based on growth suppression, no-observed-adverse-effect-levels (NOAELs) were estimated to be 1.25% (696 mg/kg/day) for males and 5% (2879 mg/kg/day) for females. As increases in serum Ca were observed in the lowest group in both sexes, a no-observed-effect level (NOEL) could not be determined in this study.

Beta-carotene and the application of transcriptomics in risk-benefit evaluation of natural dietary components.

Beta-carotene is a natural food component that is present in fruits and vegetables and is also used as a food colorant and a supplement. Beta-carotene is an anti-oxidant and a source of vitamin A. It is endowed with health beneficial properties, but a number of studies showed that with high intakes it may increase the risk for lung cancer in at risk individuals (heavy smokers, asbestos workers and alcohol users). To establish the window of benefit, it is necessary to identify early markers of effect and to obtain insight in the mechanism of action of beta-carotene, in the absence and presence of environmental risk factors. Genomics technologies are well suited to dissect the mechanisms of action and identify the markers of effect. Human cell lines can be used to analyse the effects of beta-carotene, but exposure studies with beta-carotene show that cell lines display a widely variant behaviour, which hampers translation to the in vivo situation in humans. Alternatively, animal studies can be used. Especially the ferret seems to be a good model, but little sequence information of this species is available. However, heterologous hybridization on human cDNA seems possible and provides and a new tool for molecular analysis of health effects of beta-carotene.

Toxicity of oxidized beta-carotene to cultured human cells.

Carotenoids are effective antioxidants in vitro, but they are also susceptible to autoxidation, which generates volatile and biologically active aldehydes and ketones. In a previous study, we showed that autoxidized beta-carotene inhibits Na+-K+-ATPase activity more effectively than aldehydic products derived from lipid peroxidation, such as 4-hydroxynonenal. In this study, we compared mitochondrial dysfunction in cultured human K562 erythroleukaemic and 28 SV4 retinal pigment epithelium (RPE) cells in response to the degradation products of beta-carotene autoxidation using the MTT assay. We found that oxidized beta-carotene is cytotoxic and that mitochondrial function is decreased in both K562 and RPE cells. In addition, the RPE cells were more resistant to this form of oxidative stress, suggesting that its cytotoxicity may depend on cellular antioxidant capacity.

A 90-day oral toxicity study of beta-carotene derived from Blakeslea trispora, a natural food colorant, in F344 rats.

A subchronic oral toxicity study of beta-carotene derived from Blakeslea trispora, a natural food colorant, was performed with groups of 10 male and 10 female F344 rats fed the agent at dietary levels of 0%, 0.2%, 1.0% and 5.0% for 90 days. There were no treatment-related adverse effects with regard to body weight, food and water consumption, urinalysis, ophthalmology, hematology, serum biochemistry, and organ weight data. On clinical observation, red coloring of fur was noted in both sexes of the 1.0% and 5.0% group rats, with red feces observed in all treated group animals, and necropsy revealed all rats of the treated groups to have reddish coloration of the contents of the gastro-intestinal tract, due to the pigmentation and thus lacking toxicological significance. On histopathological examination, sporadic spontaneous lesions known to occur in this strain of rats were the only findings, with no specific relation to the test substance. Thus, the no-observed-adverse-effect-level (NOAEL) was judged to be a dietary level of at least 5.0% (3127 mg/kg body weight/day for males, 3362 mg/kg body weight/day for females) for beta-carotene derived from B. trispora under the present experimental conditions.

Molecular mechanisms of toxicity of important food-borne phytotoxins.

At present, there is an increasing interest for plant ingredients and their use in drugs, for teas, or in food supplements. The present review describes the nature and mechanism of action of the phytochemicals presently receiving increased attention in the field of food toxicology. This relates to compounds including aristolochic acids, pyrrolizidine alkaloids, beta-carotene, coumarin, the alkenylbenzenes safrole, methyleugenol and estragole, ephedrine alkaloids and synephrine, kavalactones, anisatin, St. John's wort ingredients, cyanogenic glycosides, solanine and chaconine, thujone, and glycyrrhizinic acid. It can be concluded that several of these phytotoxins cause concern, because of their bioactivation to reactive alkylating intermediates that are able to react with cellular macromolecules causing cellular toxicity, and, upon their reaction with DNA, genotoxicity resulting in tumors. Another group of the phytotoxins presented is active without the requirement for bioactivation and, in most cases, these compounds appear to act as neurotoxins interacting with one of the neurotransmitter systems. Altogether, the examples presented illustrate that natural does not equal safe and that in modern society adverse health effects, upon either acute or chronic exposure to phytochemicals, can occur as a result of use of plant- or herb-based foods, teas, or other extracts.

Cytotoxic and genotoxic effects of beta-carotene breakdown products on primary rat hepatocytes.

According to Siems and colleagues, free radical attack on beta-carotene results in the formation of high amounts of cleavage products with prooxidant activities towards subcellular organelles such as mitochondria. This finding may be an explanation for the contradictory results obtained with beta-carotene in clinical efficacy and cancer prevention trials. Since primary hepatocytes proved to be very sensitive indicators of the genotoxic action of suspect mutagens/carcinogens we therefore investigated a beta-carotene cleavage products mixture (CP), apo8'- carotenal (apo8') and beta-carotene utilizing primary cultures of rat hepatocytes. The end-points tested were: the mitotic index, the percentage of necrotic and apoptotic cells, micronucleated cells, chromosomal aberrations and sister chromatid exchanges (SCE). Our results indicate a genotoxic potential of both CP and apo8' already at the concentrations 100 nM and 1 microM, i.e. at pathophysiologically relevant levels of beta-carotene and beta-carotene breakdown products. A 3 h treatment with CP induced statistically significant levels of micronuclei at concentrations of 0.1, 1 and 10 microM and chromosomal aberrations at concentrations of 1, 5 and 10 microM. Apo8' induced statistically significant levels of micronuclei at concentrations of 0.1, 1 and 5 microM and chromosomal aberrations at concentrations of 0.1, 1 and 10 microM. Statistically significant increases in SCE induction were only observed at a concentration of 10 microM CP and apo8'. In contrast, no significant cytotoxic effects of these substances were observed. Since beta-carotene induced neither significant cytotoxic nor genotoxic effects at concentrations ranging from 0.01 up to 10 microM, these observations indicate that most likely beta-carotene breakdown products are responsible for the occurrence of carcinogenic effects found in the Alpha-Tocopherol Beta-Carotene Cancer Prevention (ATBC) Study and the Beta-CArotene and RETinol Efficacy Trial (CARET).

Effect of beta-carotene supplementation on rats submitted to chronic ethanol ingestion.

Chronic alcohol consumption is directly related to the induction of liver damage. The continuous use of ethanol induces the isoenzyme cytochrome P450CYP2E1, which promotes the formation of free radicals, resulting in lipid peroxidation. Among the main antioxidants are vitamin E, reduced glutathione (GSH), vitamin C, and beta-carotene. beta-Carotene has antioxidant activity per se, with a probable protective effect against different types of cancer. However, some studies have shown an increased number of cancer cells when beta-carotene is administered in the presence of chronic ethanol ingestion. On this basis, the objective of the present study was to assess the effect of beta-carotene supplementation on rats chronically treated with a hydroalcoholic solution by determining the levels of vitamin E, beta-carotene, GSH, and thiobarbituric acid reactive species (TBARS). Both the plasma and liver concentrations of beta-carotene were higher in the supplemented groups. Plasma vitamin E levels were decreased in the control group and liver vitamin E levels were significantly decreased (p < 0.05) in all groups compared to basal levels. GSH levels were increased over basal values in the group supplemented with beta-carotene for 14 days. TBARS values were increased as much as four-fold in the control group at 14 days, and declined again at 28 days, whereas they were increased in the supplemented group, with the increase remaining until the end of the experiment. The study indicates that beta-carotene had no beneficial effect as an antioxidant on rats submitted to chronic alcohol intake, and could be act as prooxidant when administered with ethanol.