Genotoxicity evaluation of environmental pollutants using analysis of nucleolar alterations.

Nucleolar alterations resulting from the action of either chemical or physical agents can serve as important genotoxicity biomarkers. In this study, the efficiency of AgNOR banding technique to identify the presence of nucleoli in micronucleus and assess nucleolar alterations in aberrant cells of Allium cepa was evaluated. Seeds of this plant were exposed to both water samples from a river that receives untreated urban effluent and to the trifluralin herbicide (0.84 mg/L concentration), both analyzed in two different seasons (summer and winter seasons). Samples induced significant frequencies of chromosomal and nuclear aberrations and micronuclei, as observed in cells submitted to conventional chromosomal staining. The herbicide caused a significant increase in the number of nucleoli and micronuclei, interpreted as due to the elimination of excessive nucleolar material resulting from polyploidization. The use of the AgNOR technique enabled the identification of both the presence of the nucleolus in some micronuclei and the nucleolar organizer region (NOR) behavior of aberrant cells. The NOR-banding technique showed to be an efficient tool for studying the genotoxic effects caused by a xenobiotics and a complex environmental sample.

Hazard-ranking of agricultural pesticides for chronic health effects in Yuma County, Arizona.

With thousands of pesticides registered by the United States Environmental Protection Agency, it not feasible to sample for all pesticides applied in agricultural communities. Hazard-ranking pesticides based on use, toxicity, and exposure potential can help prioritize community-specific pesticide hazards. This study applied hazard-ranking schemes for cancer, endocrine disruption, and reproductive/developmental toxicity in Yuma County, Arizona. An existing cancer hazard-ranking scheme was modified, and novel schemes for endocrine disruption and reproductive/developmental toxicity were developed to rank pesticide hazards. The hazard-ranking schemes accounted for pesticide use, toxicity, and exposure potential based on chemical properties of each pesticide. Pesticides were ranked as hazards with respect to each health effect, as well as overall chronic health effects. The highest hazard-ranked pesticides for overall chronic health effects were maneb, metam-sodium, trifluralin, pronamide, and bifenthrin. The relative pesticide rankings were unique for each health effect. The highest hazard-ranked pesticides differed from those most heavily applied, as well as from those previously detected in Yuma homes over a decade ago. The most hazardous pesticides for cancer in Yuma County, Arizona were also different from a previous hazard-ranking applied in California. Hazard-ranking schemes that take into account pesticide use, toxicity, and exposure potential can help prioritize pesticides of greatest health risk in agricultural communities. This study is the first to provide pesticide hazard-rankings for endocrine disruption and reproductive/developmental toxicity based on use, toxicity, and exposure potential. These hazard-ranking schemes can be applied to other agricultural communities for prioritizing community-specific pesticide hazards to target decreasing health risk.

Effects of trifluralin on the mouse ovary.

Trifluralin, a herbicide used to protect many arable and horticultural crops, was evaluated for its potential toxicity on the mammalian ovary. To this end, adult female mice were fed or not (control) with a trifluralin-enriched diet (150 mg/kg body weight/day) during gestation and lactation. After weaning, 3-week-old female mice from either trifluralin-treated or control groups were used to evaluate whether the exposure to this herbicide in utero and during lactation could induce stress responses in the ovary. It was found that trifluralin exposure caused a significantly higher level of p53, but not of pRb, in the whole ovary, and in particular in granulosa cells. TUNEL staining showed that herbicide treatment did not increase the apoptotic index of the somatic compartment. Also oocyte fertilizability was unaffected, as metaphase II oocytes retrieved from treated mice were capable of forming male and female pronuclei after in vitro fertilization as control mice. However, trifluralin determined a slightly higher number of oocytes with cytoplasmic degeneration compared with control animals. In conclusion, our results suggest that exposure to a low trifluralin dose during pregnancy and lactation does not impair oocyte quality, but can induce a stress response in ovarian somatic cells.

Mitotic crossing-over induced by two commercial herbicides in diploid strains of the fungus Aspergillus nidulans.

Some herbicides are suspected of promoting teratogenic, carcinogenic and mutagenic events. Detection of induced mitotic crossing-over has proven to be an indirect way of testing the carcinogenic properties of suspicious substances, because mitotic crossing-over is involved in the multistep process of carcinogenesis. We examined mitotic crossing-over induced by two commercial herbicides (diuron and trifluralin) in diploid strains of Aspergillus nidulans based on the homozygotization index. Low doses (2.5 microg/mL) of diuron were sufficient to increase the mean homozygotization index in 2.1 and 11.3 times for UT448//UT196 and Dp II-I//UT196, respectively, whereas the same dose of trifluralin increased this mean only 1.2 (UT448//UT196) and 3.5 (Dp II-I//UT196) times, respectively. The lower homozygotization index value found for trifluralin could be due to its interference with mitotic crossing-over in eukaryotic cells. We concluded that the diploid Dp II-I//UT196 of A. nidulans is more sensitive to organic compounds than UT448//UT196; these compounds cause recombinational events at a greater frequency in the latter diploid. This system holds promise as an initial test for carcinogenicity of organic compounds, including herbicides.

Origin of nuclear and chromosomal alterations derived from the action of an aneugenic agent--Trifluralin herbicide.

Trifluralin is a herbicide capable of interfering in mitotic cell division due to either microtubule depolymerization or alteration in the concentration of calcium ions within the cell. The aim of this study was to investigate the effects of trifluralin in Allium cepa meristematic cells, evaluating the induction mechanisms of the chromosomal and nuclear aberrations. In this study, A. cepa root tips were submitted for 24h treatment to several concentrations of this herbicide and 48 h recovery post-treatment. The results showed that some concentrations of trifluralin can lead to a mitotic index inhibition, besides inducing chromosomal and nuclear alterations throughout the mitotic cycle. Some of the alterations found seem to be resulting from the herbicide action in different phases and in more than one consecutive cell cycle.

Toxicological study of pesticides in air and precipitations of Paris by means of a bioluminescence method.

A detailed toxicological study on several pesticides, including chlorothalonil, cyprodynil, dichlobénil, pendimethaline, trifluraline, and alpha-endosulfan, present at trace levels in air and total atmospheric precipitations of Paris is presented. The pesticides contained in the atmospheric samples, collected during sampling campaigns in February-March 2007, are identified and quantified by a high-performance liquid chromatographic (HPLC)-UV detection method. The toxicity measurements are performed by means of the Microtox bioluminescence method, based on the evaluation of the bioluminescence inhibition of the Vibrio fischeri marine bacteria at two exposure times to the pesticide solutions. The specific toxicity, corresponding to the particular toxicity of the compound under study and represented by the EC(50) parameter, is determined for these pesticides. Also, the global toxicity, which is the toxicity of all micro-pollutants present in the sample under study, is estimated for the extracts of air and atmospheric precipitation (rainwater) samples. The specific toxicities strongly vary with the nature of the pesticide, the EC(50) parameter values being comprised between 0.17 and 0.83 mg/mL and 0.15 and 0.66 mg/mL, respectively, for exposure times of 5 and 15 min. The importance of the atmospheric samples' global toxicity and the respective contribution of the toxic potency of the various pesticides contained in these samples are discussed.

Mechanism of trifluralin-induced thyroid tumors in rats.

Trifluralin, an herbicide, has been reported to cause a significant increase in thyroid follicular cell tumors in male Fischer 344 rats. This study was designed to determine the mechanism of thyroid hyperactivity after trifluralin exposure. A group of 15 male Fischer 344 rats were exposed to trifluralin-fortified (6500 ppm) diet for 2 weeks. The time weighted average daily intake of trifluralin was 441+/-77 mg/kg/day. Ten rats of the group were sacrificed and the sera analyzed for T3, T4, and TSH levels. The livers were also analyzed for selected T4-specific UGT gene expression and total UGT enzyme activity. In the trifluralin treated rats, the serum T3 and T4 levels decreased by 17% and 90%, respectively and TSH increased by 37% more than the control rats. Trifluralin-induced total hepatic UGT enzymes (2.4-fold) and mRNA expression of selected hepatic UGT isozymes (UGT1A1, 1.4-fold; UGT1A6, 6.4-fold; UGT2B1, 3.7-fold). For the remaining 5 rats in the group, bile was collected for 2 h and analyzed for free and conjugated T3 and T4. The total amount of T4 in bile more than doubled in trifluralin treated rats. Trifluralin treatment increased bile flow, caused a 3.2-fold increase in biliary elimination of conjugated T4 and 63% increase in conjugated T3. Based on these data, the decrease in total serum T3 and T4 levels in the trifluralin treated rats was due to enhanced peripheral metabolism and an increase in bile flow that results in a compensatory increase in TSH synthesis and secretion. The increased levels of TSH with chronic exposure to trifluralin would exert a continuous stimulation of the thyroid gland leading to cellular hypertrophy and proliferation predisposing to the development of follicular cell tumors in rats.

Cancer incidence among pesticide applicators exposed to trifluralin in the Agricultural Health Study.

Trifluralin, 2,6-dinitro-N,N-dipropyl-4-trifluoromethylaniline, is a 2,6-dinitro herbicide widely used to control annual grasses and broadleaf weeds in agricultural settings. The association between trifluralin use and common cancer incidence was evaluated among 50,127 private and commercial pesticide applicators in the Agricultural Health Study (AHS), a prospective cohort study of licensed pesticide applicators and their spouses in Iowa and North Carolina. Poisson regression was used to examine internal dose-response relationships, while controlling for important lifestyle factors and other agricultural exposures. Two metrics of exposure (lifetime days and intensity-weighted lifetime days) were used in exposure-response analyses with non-exposed applicators, as well as applicators in the lowest tertile of exposure, as reference groups. Incident cancers were identified through state tumor registries from enrollment in 1993 through 2002. Trifluralin exposure was not associated with cancer incidence overall among 51% of private and commercial applicators (n=25,712) who had used trifluralin. However, there was an excess of colon cancer in the exposure category of higher half of highest tertile (rate ratios (RR) of 1.76 (95% CI=1.05-2.95) using the non-exposed as a referent and 1.93 (95% CI=1.08-3.45) using those with the lowest tertile of exposure as the referent). There was also a non-significantly elevated risk for kidney cancer and bladder cancer in the highest exposure group, although only the kidney cancer finding was consistent across exposure metrics. Although there was a possible link between trifluralin exposure and colon cancer, small numbers and inconsistencies in dose-response and subgroup analyses indicate that this may be a chance finding.

Effects of the pesticides carbofuran, chlorpyrifos, dimethoate, lindane, triallate, trifluralin, 2,4-D, and pentachlorophenol on the metabolic endocrine and reproductive endocrine system in ewes.

Many pesticides are used in the agricultural environment, and some may have the potential to disrupt reproductive or endocrine function. Ewes, in separate groups of 6, received orally into their rumen either empty gelatin capsules or capsules containing chlorpyrifos (12.5 mg/kg), trifluralin (17.5 mg/kg), lindane (2.5 mg/kg), or pentachlorophenol (2 mg/kg) 2 times per week for 43 d. Dimethoate (0.2 mg/kg), carbofuran (0.30 mg/kg), 2,4-dichlorophenoxyacetic acid (10 mg/kg), or triallate (5 mg/kg) was given 3 times per week. After 36 d of treatment, blood samples were taken every 12 min for 6 h for hormone analysis. Ewes were euthanized at the end of the study for necropsy and histopathology. No overt signs of toxicity were seen, and body weight was not affected by treatment. Carbofuran caused a significant increase in serum concentrations of thyroxine compared to control ewes, but all other pesticides, except trifluralin, resulted in a marked decrease in thyroxine concentrations. Serum concentrations of cortisol were significantly increased by trifluralin and chlorpyrifos. Concentrations of insulin in serum were markedly increased in ewes given dimethoate, lindane, trifluralin, triallate, and pentachlorophenol, and concentrations of estradiol were also significantly increased in ewes given lindane and trifluralin. Mean serum concentrations of LH were markedly decreased by trifluralin, and basal LH concentrations were significantly decreased by lindane, dimethoate, and trifluralin but increased by triallate. Both pentachlorophenol and triallate caused a significant increase in severity of oviductal intraepithelial cysts in ewes. Data suggest that several currently used pesticides could influence serum concentrations of reproductive and metabolic hormones, particularly thyroxine, the major secretory product of the thyroid and a principal regulator of metabolism.

Developmental toxicity of dinitroaniline herbicides in rats and rabbits. I. Trifluralin.

The potential developmental toxicity of trifluralin was evaluated in rats and rabbits. Pregnant rats and rabbits were dosed once daily by gavage on Gestation Days 6-15 and 6-18, respectively. Doses for rats were 0, 100, 225, 475, or 1000 mg/kg; doses for rabbits were 0, 100, 225, or 500 mg/kg. Cesarean sections were performed on rats and rabbits on Gestation Days 20 and 28, respectively. In rats, maternal toxicity was indicated in the 475 and 1000 mg/kg treatment groups by depression of body weights and food consumption. Fetal viability and morphology were not adversely affected at any dose level. Developmental toxicity was indicated at the 1000-mg/kg dose level by depression of fetal weight. The NOAEL for maternal toxicity in the rat was 225 mg/kg; the NOAEL for developmental toxicity in the rat was 475 mg/kg. In rabbits, maternal toxicity was indicated at the 225 and 500 mg/kg dose levels by abortions and/or deaths in conjunction with anorexia and cachexia. Developmental toxicity was indicated at the 500 mg/kg dose level by depressed fetal viability and weight. Fetal morphology was not adversely affected at any dose level. The NOAELs for maternal and developmental toxicity in the rabbit were 100 and 225 mg/kg, respectively. Based on these data, trifluralin did not exhibit selective toxicity toward the developing conceptus.

Mutagenicity testing of nine herbicides and pesticides currently used in agriculture.

Nine herbicides and pesticides were tested for their mutagenicity using the Drosophila sex-linked recessive lethal mutation assay. These are Ambush, Treflan, Blazer, Roundup, 2,4-D Amine, Crossbow, Galecron, Pramitol, and Pondmaster. All of these are in wide use at present. Unlike adult feeding and injection assays, the larvae were allowed to grow in medium with the test chemical, thereby providing long and chronic exposure to the sensitive and dividing diploid cells, i.e., mitotically active spermatogonia and sensitive spermatocytes. All chemicals induced significant numbers of mutations in at least one of the cell types tested. Some of these compounds were found to be negative in earlier studies. An explanation for the difference in results is provided. It is probable that different germ cell stages and treatment regimens are suitable for different types of chemicals. larval treatment may still be valuable and can complement adult treatment in environmental mutagen testing.

Herbicides to curb human parasitic infections: in vitro and in vivo effects of trifluralin on the trypanosomatid protozoans.

Leishmaniasis is a major tropical disease for which current chemotherapies, pentavalent antimonials, are inadequate and cause severe side effects. It has been reported that trifluralin, a microtubule-disrupting herbicide, is inhibitory to Leishmania amazonensis. In this study, the in vitro effect of trifluralin on different species of trypanosomatid protozoans was determined. In addition to L. amazonensis, trifluralin is effective against Leishmania major and Leishmania tropica, which cause cutaneous infections, Leishmania donovani, which causes visceral disease, Leishmania panamensis, which may cause mucocutaneous infection, and Trypanosoma brucei, an important human and veterinary pathogen. Moreover, most encouragingly, trifluralin is effective in vivo as a topical ointment against L. major and Leishmania mexicana murine cutaneous leishmaniasis. Thus, trifluralin is a promising lead drug for several related, prevalent tropical diseases: leishmaniasis, trypanosomiasis of animals, and, possibly, African trypanosomiasis in humans.

Toxicology and hazard potential of trifluralin.

This paper reviews the results of toxicity studies conducted in laboratory animals to evaluate the safety of the herbicide trifluralin (TFL). The data show that TFL is slightly toxic following single oral exposure. Testing for embryotoxicity in rats and rabbits indicated no teratogenic potential, and many different mutagenicity tests showed that TFL was non-genotoxic. Subchronic and chronic toxicity testing in rats, mice and dogs indicated that TFL was haematotoxic (anaemia and methaemoglobinaemia), particularly in the dog, and slightly hepatotoxic. No-observed-effect levels of 4.8 and 41 mg/kg body weight/day, respectively, were determined in dogs and rats exposed chronically to TFL. Oncogenicity studies in rats and mice revealed no carcinogenic potential. Since the data for TFL indicated no mutagenic or other special toxicological risks, it is suggested that a safety factor of 100 could be used for the determination of the acceptable daily intake of TFL, which would be 0.05 mg/kg body weight/day.

Oncogenicity study of trifluralin in B6C3F1 mice.

B6C3F1 mice were maintained for 24 months on diets containing 0, 563, 2250 or 4500 ppm trifluralin. These dietary concentrations corresponded to daily doses of approximately 70, 285 or 570 mg/kg body weight, respectively. The control group contained 120 mice/sex and treated groups consisted of 80 mice/sex. There were no treatment-related effects on the survival, appearance or behaviour of the mice. Survival at test termination was at least 67% in each group. Compared with controls, mean body weight was significantly reduced in a dose-related manner in mice of both sexes given the 2250 and 4500 ppm diets. At 21 months, the reduction in body weight was greater than or equal to 15 and greater than or equal to 30%, respectively. At study termination, dose-related decreases in erythrocytic and leucocytic values were also observed at dietary levels of 2250 and 4500 ppm. In clinical chemistry evaluations, blood urea nitrogen levels and alkaline phosphatase activity in mice of both sexes were significantly increased at trifluralin levels of 2250 and 4500 ppm. Blood urea nitrogen also showed a marginal increase in females given the low dose of trifluralin. Alanine aminotransferase activity was significantly increased in males at all treatment levels. Although there were a number of absolute and relative organ weight changes in all three treatment groups that were significantly different from the control values, the reduced relative kidney weights in males and the increased relative liver weights in both sexes at dietary levels of 2250 and 4500 ppm were the only changes that could be correlated with altered clinical chemistry values.(ABSTRACT TRUNCATED AT 250 WORDS)

Usefulness of Cyprinodon variegatus and Fundulus grandis in carcinogenicity testing: advantages and special problems.

Cyprinodon variegatus and Fundulus grandis, two species of cyprinodontid fishes studied extensively and used in toxicological and biological investigations, are compared as laboratory test animals. Their ecology, general biology, and suitability for various types of experimentation are examined. A laboratory system for exposure of these species at critical life stages (embryos, fry, juveniles) to suspect carcinogens is described. A discussion of the use, findings, and potential of these species in oncological studies and carcinogen assays is presented, particularly in regard to responses to 3 known or suspect carcinogens (trifluralin, benzidine, and aflatoxin). Finally, advantages and disadvantages or special problems in using the species as carcinogen test animals are reviewed.

[Toxicologic characteristics of several pesticides and standards for them in water]. / Toksikologichna kharakteristika na niakoi pestitsidi i normiraneto im vuv vodite.

The progressive increase of production and application of chemicals for plant protection transmuted the problem of protection of water cleanliness into an international and national problem. The cases with incidental pesticide pollution of water-reservoirs became more numerous. A necessity arose of the supplement of our sanitary legislation with hygienic standards (MAC) for the water for the newly implemented chemicals for plant protection in the country. During the last several years, some of the most frequently used and formulated in our country pesticides were experimentally studied: Afalon and Patoran (from chemical group of carbamates and dithiocarbamates) and amide herbicide: Dimid, Ramrod, Lasso, Treflan. Complex hygienic-toxicological studies were carried out in order to study the toxicological effect of the pesticides mentioned and to elaborate hygienic norms (MAC) of the same in the water. The effect on the organoleptic qualities of water was determined as well as on the sanitary regime of waterreservoirs (according to BON5, sanitary-indictory microflora and toxicity for fishes). Toxicological studies were performed under the conditions of acute, subacute and chronic experiment. The results obtained allowed the hygienic-toxicological characteristics and the proposal of hygienic standards (MAC) for the pesticides studied in water.

The effects of five weeks treatment with dinitro-o-cresol- or trifluralin-containing pesticides on the germ cells of male mice.

The effects of two pesticides, the insecticide-herbicide Krezonit E, which contains 50% dinitro-o-cresol, and the herbicide Olitref, which contains 26% trifluralin (2,6-dinitro-N,N-dipropyl-4-trifluoromethylaniline), on the gonads and germ cells of male mice were studied. The pesticides were given twice a week for 5 weeks in i.p. doses of 0.6 mg kg-1 for Krezonit E and 6.0 mg kg-1 for Olitref. These doses are 1% of the i.p. LD50. Cytogenic analysis of germ cells carried out from 3 weeks onwards after the last treatment day showed that Olitref significantly increased the frequency of germinal chromosomal abnormalities at 6-7 weeks after treatment. This pesticide increased the frequency of autosomal univalents X/Y separations at meiotic metaphase and multivalent configurations. Krezonit E did not increase significantly the number of chromosomal abnormalities, although there was some increase at 3 weeks, mainly in the form of autosomal univalents.