Biomarkers and bioindicators of the environmental condition using a fish species (Pimelodus maculatus Lacepède, 1803) in a tropical reservoir in Southeastern Brazil / Biomarcadores e bioindicadores da condição ambiental usando uma espécie de peixe (Pimelodus maculatus Lacepède, 1803) em um reservatório tropical no Sudeste do Brasil

Abstract The Funil Reservoir receives a large amount of xenobiotics from the Paraíba do Sul River (PSR) from large number of industries and municipalities in the watershed. This study aimed to assess environmental quality along the longitudinal profile of the Paraíba do Sul River-Funil Reservoir system, by using biomarkers and bioindicators in a selected fish species. The raised hypothesis is that Funil Reservoir acts as a filter for the xenobiotics of the PSR waters, improving river water quality downstream the dam. Two biomarkers, the ethoxyresorufin-O-deethylase activity (EROD), measured as fluorimetricly in S9 hepatic fraction, and the micronuclei frequency (MN), observed in erythrocytes of the cytoplasm, and three bioindicators, the hepatosomatic index (HSI), gonadosomatic index (GSI) and condition factor (CF) were used in Pimelodus maculatus, a fish species widely distributed in the system. Four zones were searched through a longitudinal gradient: 1, river upstream from the reservoir; 2, upper reservoir; 3, lower reservoir; 4, river downstream of the reservoir. EROD activity and HSI and GSI had significant differences among the zones (P<0.05). The upper reservoir had the lowest EROD activity and HSI, whereas the river downstream of the reservoir had the highest EROD and lowest GSI. The river upstream from the reservoir showed the highest HSI and GSI. It is suggested that the lowest environmental condition occur at the river downstream of the reservoir, where it seems to occur more influence of xenobiotics, which could be associated with hydroelectric plant operation. The hypothesis that Funil reservoir acts as a filter decanting pollution from the Paraíba do Sul River waters was rejected. These results are novel information on this subject for a native fish species and could be useful for future comparisons with other environments.

Resumo O reservatório do Funil recebe uma grande quantidade de contaminantes xenobióticos do Rio Paraíba do Sul (RPS) provenientes de grandes indústrias e municípios situados na bacia hidrográfica. O objetivo deste estudo é avaliar a qualidade ambiental ao longo de um perfil longitudinal do sistema rio Paraíba do Sul-reservatório do Funil, através de biomarcadores e bioindicadores em uma espécie de peixe selecionada. A hipótese a ser testada é de que o reservatório do Funil funciona como filtro para poluentes xenobioticos de águas do rio Paraíba do Sul, melhorando a qualidade da água à jusante da represa. Foram usados dois biomarcadores: a atividade de etoxiresorufina-O-desetilase (EROD), medida fluorimetricamente na fração S9 hepática, e a Freqüência de Micronúcleos (MN), observada no citoplasma dos eritrócitos; e também três bioindicadores: Índice hepato-somático (IHS), Índice gonado-somático (IGS) e Fator de Condição (FC) em Pimelodus maculatus, uma espécie de peixe amplamente distribuída no sistema. Quatro zonas foram amostrados ao longo do gradiente longitudinal: 1, rio a montante do reservatório; 2, parte superior do reservatório; 3, parte inferior do reservatório; 4, rio à jusante do reservatório. A atividade de EROD, o IHS e o IGS apresentaram diferenças significativas (P<0.05) entre as zonas. A atividade EROD e o IHS foram mais baixos na parte superior do reservatório, enquanto que no rio à jusante do reservatório, a atividade de EROD foi mais alta e o IGS foi mais baixo. O rio acima do reservatório apresentou maiores IHS e IGS. É sugerido que a pior condição ambiental ocorreu no rio abaixo do reservatório, o que poderia ser associado às influências das operações da usina hidroelétrica. A hipótese de que o reservatório do Funil atue como filtro decantando a poluição do rio Paraíba do Sul foi rejeitada. Estes dados são novas informações sobre este tema para uma espécie nativa e podem ser úteis para futuras comparações outros ambientes.

Biomarkers and bioindicators of the environmental condition using a fish species (Pimelodus maculatus Lacepède, 1803) in a tropical reservoir in Southeastern Brazil.

The Funil Reservoir receives a large amount of xenobiotics from the Paraíba do Sul River (PSR) from large number of industries and municipalities in the watershed. This study aimed to assess environmental quality along the longitudinal profile of the Paraíba do Sul River-Funil Reservoir system, by using biomarkers and bioindicators in a selected fish species. The raised hypothesis is that Funil Reservoir acts as a filter for the xenobiotics of the PSR waters, improving river water quality downstream the dam. Two biomarkers, the ethoxyresorufin-O-deethylase activity (EROD), measured as fluorimetricly in S9 hepatic fraction, and the micronuclei frequency (MN), observed in erythrocytes of the cytoplasm, and three bioindicators, the hepatosomatic index (HSI), gonadosomatic index (GSI) and condition factor (CF) were used in Pimelodus maculatus, a fish species widely distributed in the system. Four zones were searched through a longitudinal gradient: 1, river upstream from the reservoir; 2, upper reservoir; 3, lower reservoir; 4, river downstream of the reservoir. EROD activity and HSI and GSI had significant differences among the zones (P<0.05). The upper reservoir had the lowest EROD activity and HSI, whereas the river downstream of the reservoir had the highest EROD and lowest GSI. The river upstream from the reservoir showed the highest HSI and GSI. It is suggested that the lowest environmental condition occur at the river downstream of the reservoir, where it seems to occur more influence of xenobiotics, which could be associated with hydroelectric plant operation. The hypothesis that Funil reservoir acts as a filter decanting pollution from the Paraíba do Sul River waters was rejected. These results are novel information on this subject for a native fish species and could be useful for future comparisons with other environments.

Effects of Euphorbia milii latex on mitogen-induced lymphocyte proliferation / Efeito do latex de Euphorbia milii sobre a proliferação de linfócitos induzida por mitogénio

The crude latex of "Crown-of-Thorns" (Euphorbia milii var hislopii, syn E.splendens) is a potent plant molluscicide. For this reason, toxicological studies have been performed to evaluate the health risks posed by its use in schistosomiasis control programs. The present study is part of a more comprehensive immunotoxicological evaluation of this molluscicide. Here, we investigated the effects of E. milii latex on the proliferation of human lymphocytes in vitro. Lyophilized latex of E. milii (0, 0.5, 5, 25 and 50 µg/ml) was incubated with whole blood in the presence of proliferation stimulators, i.e. lectins (phytohemagglutinin, concanavalin A and pokeweed mitogen), as well as with human monoclonal antibody against CD3 and tetanus toxoid. Cell proliferation was measured by ³H-thymidine incorporation, and the effects of latex on mitogen-induced cell proliferation were compared to the effects of 10 ng/ml of 12-O-tetradecanoylphorbol-13-acetate (TPA). Results showed that mitogen-induced cell proliferation was markedly enhanced by E. milii latex. This synergistic effect of latex on mitogen-induced lymphocyte proliferation may be due to the presence of TPA-like phorbol esters and/or to mitogenic plant lectins.

O látexbrutoda "Coroa de Cristo" (Euphorbia miliivarhislopii, syn E.splendens) é um potente moluscicidavegetal. Neste sentido, são necessários estudos toxicológicosque visemavaliar possíveis riscos à saúdeassociados ao uso em larga escala desta espécie em áreas endêmicas para esquistossomose. O presente estudo é parte deuma avaliação mais abrangentesobre o potencial tóxico destemoluscicida. Foram investigados in vitro osefeitos dolátex da E.miliisobre a proliferação delinfócitoshumanos. O látexliofilizado (0; 0,5;5;25 e 50 µg/ml)foi incubado comsangue totalna presençade agentes mitogênicos, tais como lectinas(fitohemaglutinina, concanavalina Ae pokeweed), anticorpomonoclonalhumano anti-CD3etoxóide tetânico. A proliferação celularfoi quantificada atravésincorporaçãode ³H-timidina eos efeitos do látexnaproliferação celular induzida por agentes mitogênicosforam comparados comos efeitos de10 ng/mlde12-O-tetradecanoilforbol-13-acetato (TPA). Os resultados demonstram quea proliferação celular induzida poragentes mitogênicosfoimarcadamenteaumentada na presença do látex daE.milii.Oefeito sinérgico observado pode ser devidoà presença deésteres de forbol, como o TPA, e/oude lectinas com ação mitogênica presentes nesta espécie vegetal.

Evaluation of the genotoxicity of Orthosiphon stamineus aqueous extract.

AIM OF THE STUDY: Orthosiphon stamineus, Benth, also known as Misai Kucing in Malaysia and Java tea in Indonesia, is traditionally used in Southeastern Asia to treat kidney dysfunctions, diabetes, gout and several other illnesses. Recent studies of Orthosiphon stamineus pharmacological profile have revealed antioxidant properties and other potentially useful biological activities thereby lending some scientific support to its use in folk medicine. So far the genotoxicity of Orthosiphon stamineus extracts has not been evaluated. In this study the genotoxic potential of Orthosiphon stamineus aqueous extract was investigated by the Salmonella/microsome mutation assay and the mouse bone marrow micronucleus test. MATERIALS AND METHODS: Chemical composition of Orthosiphon stamineus aqueous extract was analyzed by High Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD). The Salmonella/microsome assay (TA97a, TA98, TA100 and TA1535; plate incorporation method) was performed in the presence or in the absence of extrinsic metabolic activation (S9 mixture). In the mouse micronucleus assay, Orthosiphon stamineus extract was administered by gavage (0, 500, 2000 and 4000 mg/kg body weight/day for 3 days) to male and female Swiss Webster mice (N=6 per dose per sex) and bone marrow cells were harvested 24 h after the last dose. Ethoxy-resorufin-O-dealkylase (EROD) and benzyloxy-resorufin-O-dealkylase (BROD) activities were determined in mouse liver microsomes. RESULTS: The chemical analysis revealed that the Orthosiphon stamineus extract contained flavonoids (sinensetin, eupatorin), caffeic acid, and rosmarinic acid (44.00±1.879 µg/mg), the latter seemed to be one of its major constituent. Tested at doses up to 5000 µg/plate, the Orthosiphon stamineus extract was not toxic to Salmonella tester strains and did not increase the number of revertant colonies over the background incidence. In the mouse bone marrow assay, the extract did not alter the polychromatic:normochromatic erythrocytes (PCE:NCE) ratio, nor did it increase the incidence of micronucleated polychromatic erythrocytes (MNPEs). No overt toxicity and no change of CYP1A (EROD) and 2B9/10 (BROD) activities were noted. CONCLUSIONS: Based on the aforementioned findings, it is concluded that the use of Orthosiphon stamineus in the traditional medicine poses no genotoxic risk.

Comparative efficacies of two antimony regimens to treat Leishmania braziliensis-induced cutaneous Leishmaniasis in rhesus macaques (Macaca mulatta).

This study compared the efficacies of two N-methylglucomine antimoniate (MA) dose regimens for treating macaques with Leishmania braziliensis-induced chronic skin disease. Whereas all animals treated with the full dose (20 mg MA/kg/day) were cured, 50% of the monkeys receiving a low-dose regimen (5 mg MA/kg/day) relapsed. The antimony concentrations in macaque plasma and tissue samples were greater in the full-dose group than in that receiving a subtherapeutic MA regimen. Our data also suggest the presence of drug-induced hepatic pathology.

Lack of constitutive and inducible ethoxyresorufin-O-deethylase activity in the liver of suckermouth armored catfish (Hypostomus affinis and Hypostomus auroguttatus, Loricariidae).

We investigated the presence and inducibility of CYP1A in suckermouth catfish (Hypostomus affinis and Hypostomus auroguttatus, Loricariidae), tilapia (Oreochromis niloticus, Cichlidae) and mice (Mus musculus, Muridae). Alkoxyresorufin-O-dealkylases (EROD, MROD, PROD and BROD) were detected and proved to be inducible (beta-naphthoflavone, BNF or dimethylbenz[a]anthracene, DMBA, 50 mg/kg bw ip) in liver microsomes from tilapia and mice. In loricariids, alkoxyresorufin-O-dealkylases were either undetectable (MROD/EROD) or very low (PROD/BROD), and so they remained after treatment with BNF or DMBA. Ethoxycoumarin-O-deethylase (ECOD) was recorded in all species and proved not to be inducible by BNF or DMBA. In loricariids and tilapia, ECOD was not depressed by a concentration of alpha-naphthoflavone (CYP1A-inhibitor) that markedly depressed EROD in tilapia. A CYP1A-like protein was detected by a monoclonal antibody in rats, mice and tilapia, but not in loricariids. A polyclonal antibody, however, detected a CYP1A-like protein in liver microsomes of loricariids. Suckermouth catfish, rats, mice and tilapia express a protein reactive with a polyclonal antibody against trout CYP3A. Loricariids and tilapia exhibited marked genotoxic responses (enhanced incidence of micronucleated erythrocytes) following treatment DMBA (50 mg/kg bw ip), a promutagen activated by CYP1A/1B. Therefore, although not exhibiting EROD, a CYP1A-mediated activity, loricariids converted DMBA into its genotoxic metabolites. Our findings suggest that the CYP1A-like protein of locariid catfish recognizes DMBA, but not ethoxyresorufin, as a substrate.

Activity of liver microsomal enzymes during the chronic phase of murine schistosomiasis

The effects of schistosomiasis on microsomal enzymes were studied on post-infection day 90 when accumulated damage and fibrosis are most intense but granulomatous reaction around the eggs harbored in the liver is smaller than during the earlier phases. Swiss Webster (SW) and DBA/2 mice of either sex (N = 12 per sex per group) were infected with 100 Schistosoma mansoni cercariae on postnatal day 10 and killed on post-infection day 90. Cytochrome P-450 (CYP) concentration and alkoxyresorufin-O-dealkylases (EROD, MROD, BROD, and PROD), p-nitrophenol-hydroxylase (PNPH), coumarin-7-hydroxylase (COH), and UDP-glucuronosyltransferase (UGT) activities were measured in hepatic microsomes. Age-matched mice of the same sex and strain were used as controls. In S. mansoni-infected mice, CYP1A- and 2B-mediated activities (control = 100 percent) were reduced in SW (EROD: male (M) 36 percent, female (F) 38 percent; MROD: M 38 percent, F 39 percent; BROD: M 46 percent, F 19 percent; PROD: M 50 percent, F 28 percent) and DBA/2 mice (EROD: M 64 percent, F 58 percent; MROD: M 60 percent; BROD: F 49 percent; PROD: M 73 percent) while PNPH (CYP2E1) was decreased in SW (M 31 percent, F 38 percent) but not in DBA/2 mice. COH did not differ between infected and control DBA/2 and UGT, a phase-2 enzyme, was not altered by infection. In conclusion, chronic S. mansoni infection reduced total CYP content and all CYP-mediated activities evaluated in SW mice, including those catalyzed by CYP2E1 (PNPH), CYP1A (EROD, MROD) and 2B (BROD, PROD). In DBA/2 mice, however, CYP2A5- and 2E1-mediated activities remained unchanged while total CYP content and activities mediated by other CYP isoforms were depressed during chronic schistosomiasis.

Activity of liver microsomal enzymes during the chronic phase of murine schistosomiasis.

The effects of schistosomiasis on microsomal enzymes were studied on post-infection day 90 when accumulated damage and fibrosis are most intense but granulomatous reaction around the eggs harbored in the liver is smaller than during the earlier phases. Swiss Webster (SW) and DBA/2 mice of either sex (N = 12 per sex per group) were infected with 100 Schistosoma mansoni cercariae on postnatal day 10 and killed on post-infection day 90. Cytochrome P-450 (CYP) concentration and alkoxyresorufin-O-dealkylases (EROD, MROD, BROD, and PROD), p-nitrophenol-hydroxylase (PNPH), coumarin-7-hydroxylase (COH), and UDP-glucuronosyltransferase (UGT) activities were measured in hepatic microsomes. Age-matched mice of the same sex and strain were used as controls. In S. mansoni-infected mice, CYP1A- and 2B-mediated activities (control = 100%) were reduced in SW (EROD: male (M) 36%, female (F) 38%; MROD: M 38%, F 39%; BROD: M 46%, F 19%; PROD: M 50%, F 28%) and DBA/2 mice (EROD: M 64%, F 58%; MROD: M 60%; BROD: F 49%; PROD: M 73%) while PNPH (CYP2E1) was decreased in SW (M 31%, F 38%) but not in DBA/2 mice. COH did not differ between infected and control DBA/2 and UGT, a phase-2 enzyme, was not altered by infection. In conclusion, chronic S. mansoni infection reduced total CYP content and all CYP-mediated activities evaluated in SW mice, including those catalyzed by CYP2E1 (PNPH), CYP1A (EROD, MROD) and 2B (BROD, PROD). In DBA/2 mice, however, CYP2A5- and 2E1-mediated activities remained unchanged while total CYP content and activities mediated by other CYP isoforms were depressed during chronic schistosomiasis.

Relationships between fetal body weight of Wistar rats at term and the extent of skeletal ossification.

We investigated the relationship between fetal body weight at term (pregnancy day 21) and the extent of ossification of sternum, metacarpus, metatarsus, phalanges (proximal, medial and distal) of fore- and hindlimbs and cervical and coccygeal vertebrae in Wistar rats. The relationships between fetal body weight and sex, intrauterine position, uterine horn, horn size, and litter size were determined using historical control data (7594 fetuses; 769 litters) of untreated rats. Relationships between body weight and degree of ossification were examined in a subset of 1484 historical control fetuses (154 litters) which were subsequently cleared and stained with alizarin red S. Fetal weight was independent of horn size, uterine horn side (left or right) or intrauterine position. Males were heavier than females and fetal weight decreased with increasing litter size. Evaluation of the skeleton showed that ossification of sternum, metacarpus and metatarsus was extensively complete and independent of fetal weight on pregnancy day 21. In contrast, the extent of ossification of fore- and hindlimb phalanges and of cervical and sacrococcygeal vertebrae was dependent on fetal body weight. The strongest correlation between body weight and degree of ossification was found for hindlimb, medial and proximal phalanges. Our data therefore suggest that, in full-term rat fetuses (day 21), reduced ossification of sternum, metacarpus and metatarsus results from a localized impairment of bone calcification (i.e., a malformation or variation) rather than from general growth retardation and that ossification of hindlimb (medial and proximal) phalanges is a good indicator of treatment-induced fetal growth retardation.

Relationships between fetal body weight of Wistar rats at term and the extent of skeletal ossification

We investigated the relationship between fetal body weight at term (pregnancy day 21) and the extent of ossification of sternum, metacarpus, metatarsus, phalanges (proximal, medial and distal) of fore- and hindlimbs and cervical and coccygeal vertebrae in Wistar rats. The relationships between fetal body weight and sex, intrauterine position, uterine horn, horn size, and litter size were determined using historical control data (7594 fetuses; 769 litters) of untreated rats. Relationships between body weight and degree of ossification were examined in a subset of 1484 historical control fetuses (154 litters) which were subsequently cleared and stained with alizarin red S. Fetal weight was independent of horn size, uterine horn side (left or right) or intrauterine position. Males were heavier than females and fetal weight decreased with increasing litter size. Evaluation of the skeleton showed that ossification of sternum, metacarpus and metatarsus was extensively complete and independent of fetal weight on pregnancy day 21. In contrast, the extent of ossification of fore- and hindlimb phalanges and of cervical and sacrococcygeal vertebrae was dependent on fetal body weight. The strongest correlation between body weight and degree of ossification was found for hindlimb, medial and proximal phalanges. Our data therefore suggest that, in full-term rat fetuses (day 21), reduced ossification of sternum, metacarpus and metatarsus results from a localized impairment of bone calcification (i.e., a malformation or variation) rather than from general growth retardation and that ossification of hindlimb (medial and proximal) phalanges is a good indicator of treatment-induced fetal growth retardation.

Absence of tumor promoting activity of Euphorbia milii latex on the mouse back skin.

Euphorbia milii (Euphorbiaceae) is a decorative plant used in gardens and living fences. In China, it has also been employed in herbal remedies for hepatitis and abdominal edema. Since E. milii latex--lyophilized or in natura--proved to be a potent plant molluscicide, its toxicity to non-target organisms has been comprehensively studied. Concerns on a possible tumor promoting activity have discouraged its use as a locally-available alternative molluscicide in schistosomiasis control programs. Two in vitro assays (inhibition of metabolic cooperation in V79 cells and Epstein-Barr virus induction in Raji cells) had suggested that E. milii latex contained tumor-promoting substances. This study was undertaken to verify whether the latex acts as a tumor promoter in vivo as well. A single dose of the initiating agent DMBA (400 nmol) was applied on the back skin of male and female DBA/2 mice. Testing for tumor promoting activity began 10 days after initiation. Tetradecanoyl phorbol acetate (TPA) (5 nmol, positive control), lyophilized latex (20, 60 and 200 microg per mouse) or acetone (vehicle control) were applied on mouse back skin twice a week for 20 weeks. In TPA-treated mice, papillomas were firstly noted during the 11th week, and by the 17th week all animals exhibited skin tumors. No tumors developed in mice treated with the solvent alone and in those exposed to latex. Findings from the present study therefore indicated that E. milii crude latex does not act as a tumor promoting agent on the mouse back skin assay.

Inhibition of cyclophosphamide-induced teratogenesis by beta-ionone.

Beta-ionone (BI) is a degraded (C 13) sesquiterpene found in plant essential oils. It has been used in the synthesis of perfume chemicals and vitamin A. Recently, it was reported that BI is a rather potent in vitro inhibitor of CYP2B1-catalysed reactions in rat liver microsomes. The present study was performed to investigate whether inhibition of CYP2B1 reactions by BI could lead to an attenuation of cyclophosphamide (CP)-induced embryotoxicity in the rat. In a preliminary experiment, a dose-dependent prolongation of pentobarbital sleeping time in male and female Wistar rats suggested that BI inhibits CYP2B1 in vivo as well. In a second experiment, rats were treated by gavage with BI (0, 250, 500, 750 or 1000 mg/kg body wt) 45 min prior to a subcutaneous injection of either CP (7.5 mg/kg body wt) or its vehicle (saline) on day 11 of pregnancy. BI alone, at the highest dose tested, caused a high proportion of resorptions. Lower doses of BI, however, clearly attenuated CP-induced embryolethality and teratogenicity. These results seem to support the view that, as far as rats are concerned, CYP2B1 plays an important role in the conversion of CP into its embryolethal and teratogenic metabolites.

Induction of liver monooxygenases by annatto and bixin in female rats.

Annatto or urucum is an orange-yellow dye obtained from Bixa orellana seeds. It has been used as a natural dye in a variety of food products, drugs and cosmetics, and also in Brazilian cuisine as a condiment ('colorau'). Bixin, a carotenoid devoid of provitamin A activity, is the main pigment found in annatto. Some carotenoids (canthaxanthin, astaxanthin and beta-Apo-8'-carotenal) are known to be potent inducers of CYP1A1, a property not shared by others (beta-carotene, lycopene and lutein). Little is known, however, about the CYP1A1-inducing properties of bixin and annatto. The present study was performed to determine the effects of an annatto extract (28% bixin) and bixin (95% pure) on rat liver monooxygenases. Adult female Wistar rats were treated by gavage with daily doses of annatto (250 mg/kg body weight, which contains approximately 70 mg bixin/kg body weight), bixin (250 mg/kg body weight) or the vehicle only (corn oil, 3.75 g/kg body weight) for 5 consecutive days, or were not treated (untreated control). The activities of aniline-4-hydroxylase (A4H), ethoxycoumarin-O-deethylase (ECOD), ethoxy- (EROD), methoxy- (MROD), pentoxy- (PROD) and benzyloxy- (BROD) resorufin-O-dealkylases were measured in liver microsomes. Annatto (250 mg/kg containing 70 mg bixin/kg) induced EROD (3.8x), MROD (4.2x), BROD (3.3x) and PROD (2.4x). Bixin (250 mg/kg) was a weaker inducer of EROD (2.7x), MROD (2.3x) and BROD (1.9x) and did not alter PROD, A4H or ECOD activities. These results suggest that constituents of the extract other than bixin play an important role in the induction of CYP1A and CYP2B observed with annatto food colorings.

Induction of liver monooxygenases by annatto and bixin in female rats

Annatto or urucum is an orange-yellow dye obtained from Bixa orellana seeds. It has been used as a natural dye in a variety of food products, drugs and cosmetics, and also in Brazilian cuisine as a condiment ('colorau'). Bixin, a carotenoid devoid of provitamin A activity, is the main pigment found in annatto. Some carotenoids (canthaxanthin, astaxanthin and á-Apo-8'-carotenal) are known to be potent inducers of CYP1A1, a property not shared by others (á-carotene, lycopene and lutein). Little is known, however, about the CYP1A1-inducing properties of bixin and annatto. The present study was performed to determine the effects of an annatto extract (28 percent bixin) and bixin (95 percent pure) on rat liver monooxygenases. Adult female Wistar rats were treated by gavage with daily doses of annatto (250 mg/kg body weight, which contains approximately 70 mg bixin/kg body weight), bixin (250 mg/kg body weight) or the vehicle only (corn oil, 3.75 g/kg body weight) for 5 consecutive days, or were not treated (untreated control). The activities of aniline-4-hydroxylase (A4H), ethoxycoumarin-O-deethylase (ECOD), ethoxy- (EROD), methoxy- (MROD), pentoxy- (PROD) and benzyloxy- (BROD) resorufin-O-dealkylases were measured in liver microsomes. Annatto (250 mg/kg containing 70 mg bixin/kg) induced EROD (3.8x), MROD (4.2x), BROD (3.3x) and PROD (2.4x). Bixin (250 mg/kg) was a weaker inducer of EROD (2.7x), MROD (2.3x) and BROD (1.9x) and did not alter PROD, A4H or ECOD activities. These results suggest that constituents of the extract other than bixin play an important role in the induction of CYP1A and CYP2B observed with annatto food colorings

Adverse health consequences of environmental exposure to 'Endocrine Disruptors'

During the last decade concern has grown on the possible adverse health effects of chemicals which are capa ble of interacting with the endocrine system. Concerns on the effects of 'endocrine disruptors' (EDs) are largely based on reports of adverse effects on wildlife reproduction and on the plausible hypothesis that exposure to these substances is responsible for an increased incidence of certain estrogen-sensitive types of cancer reproductive tract disorders and poor sperm quality. Most environmental EDs have proved to possess rather weak hormone-like effects, much weaker than those of physiological hormones, in in vitro and in vivo assays. Since EDs are found at relatively low levels in the environment, health risks posed by them would critically depend on the possibility of non-monotonic dose-effect relationships, on the relevance of low dose effects and on the type of interaction between different EDs...

Evaluation of the developmental toxicity of annatto in the rat.

Annatto, a dye extracted from Bixa orellana seeds, is used as a color additive in butter, cheese and in a variety of other foods as well as in drugs and cosmetics. Toxicological data on annatto and on its main carotenoid pigment bixin are still scarce. In this study we evaluated the developmental toxicity of annatto (28% of bixin). Annatto (0, 31.2, 62.5, 125, 250 and 500 mg/kg body weight/day) was given by gavage to Wistar rats on days 6-15 of pregnancy. Ceasarean sections were performed on day 21. Implantations, living and dead fetuses and resorptions were recorded. Fetuses were weighed and examined for externally-visible anomalies. One-third of fetuses from each litter was examined for visceral anomalies by a microsectioning technique. The remaining fetuses were cleared and stained with Alizarin Red S for skeleton evaluation. No adverse effect of annatto on the mothers was noted. No increase in embryolethality and no reduction of fetal body weight were observed among annatto-exposed rats. Annatto did not induce any increase in the incidence of externally-visible, visceral or skeletal anomalies in the exposed offspring. These findings suggest that annatto was neither maternally toxic nor embryotoxic in the rat. Therefore, the no-observed-adverse-effect level (NOAEL) for annatto-induced maternal and developmental toxicity was 500 mg/kg body weight/day or greater (or > or = 140 mg bixin/kg body weight/day) by the oral route.

Effects of pregnancy and protein-energy malnutrition on monooxygenase O-dealkylation activity in rat liver microsomes

Xenobiotic metabolism is influenced by a variety of physiological and environmental factors including pregnancy and nutritional status of the individual. Pregnancy has generally been reported to cause a depression of hepatic monooxygenase activities. Low-protein diets and protein-energy malnutrition have also been associated with a reduced activity of monooxygenases in nonpregnant animals. We investigated the combined effects of pregnancy and protein-energy malnutrition on liver monooxygenase O-dealkylation activity. On pregnancy day 0 rats were assigned at random to a group fed ad libitum (well-nourished, WN) or to a malnourished group (MN) which received half of the WN food intake (12 g/day). WN and MN rats were killed on days 0 (nonpregnant), 11 or 20 of pregnancy and ethoxy- (EROD), methoxy- (MROD) and penthoxy- (PROD) resorufin O-dealkylation activities were measured in liver microsomes. Only minor changes in enzyme activities were observed on pregnancy day 11, but a clear-cut reduction of monooxygenase activities (pmol resorufin min-1 mg protein-1) was noted near term (day 0 vs 20, means + or _ SD, Student t-test, P<0.05) in WN (EROD: 78.9 + or - 15.1 vs 54.6 + or - 10.2; MROD: 67.8 + or - 10.0 vs 40.9 + or - 7.2; PROD: 6.6 + or - 0.9 vs 4.3 + or - 0.8) and in MN (EROD: 89.2 + or - 23.9 vs 46.9 + or - 15.0; MROD: 66.8 + or - 13.8 vs 27.9 + or - 4.4; PROD: 6.3 + or - 1.0 vs 4.1 + or - 0.6) dams. On pregnancy day 20 MROD was lower in MN than in WN dams. Malnutrition did not increase the pregnancy-induced reduction of EROD and PROD activities. Thus, the present results suggest that the activities of liver monooxygenases are reduced in near-term pregnancy and that protein-energy malnutrition does not alter EROD or PROD in pregnant rats

Study of the effects of ß-myrcene on rat fertility and general reproductive performance

Beta-Myrcene (MYR) is a monoterpene found in the oils of a variety of aromatic plants including lemongrass, verbena, hop, bay, and others. MYR and essential oils containing this terpenoid compound are used in cosmetics, household products, and as flavoring food additives. This study was undertaken to investigate the effects of MYR on fertility and general reproductive performance in the rat. MYR (0, 100, 300 and 500 mg/kg) in peanut oil was given by gavage to male Wistar rats (15 per dose group) for 91 days prior to mating and during the mating period, as well ass to females (45 per dose group) continuously for 21 days before mating, during mating and pregnancy, and throughout the period of lactatiomn up to postnatal day 21. On day 21 of pregnancy one-third of the females of each group were submitted to cesarean section. Resorption, implantation, as well as dead and live fetuses were counted. All fetuses were examined for external malformation, weighed, and cleared and stained with Alizarin Red S for skeleton evaluation. The remaining dams were allowed to give birth to their offspring. The progeny was examined at birth and subsequently up to postnatal day 21. Mortality, weight gain and physical signs of postnatal development were evaluated. Except for an increase in liver and kidney weights, no other sign of toxicity was noted in male and female rats exposed to MYR. MYR did not affect the mating index (proportion of females impregnated by males) or the pregnancy index (ratio of pregnant to sperm-positive females). No sign of maternal toxicity and no increase in externally visible malformations were observed at any dose level. Only at the highest dose tested (500 mg/kg) did MYR induce an increase in the resorption rate and a higher frequency of fetal skeleton anomalies. No adverse effect of MYR on postnatal weight gain was noted but days of appearance of primary coat, incisor eruption and eye opening were slightly delayed in the exposed offspring. On the basis of the data presented in this paper the no-observed-adverse-effect level (NOAEL) for toxic effects on fertility and general reproductive performance can be set at 300 mg of Beta-myrcene/kg body weight by the oral route.