In vitro antiandrogenic effects of the herbicide linuron and its metabolites.

Although the herbicide linuron is banned for use in the EU due to its reproductive and developmental toxicity, it can still be found in randomly sampled foods grown in and outside the EU. It is not clear if metabolites of linuron can contribute to the endocrine disrupting effects following exposure to the parent compound. To address this gap, we analysed linuron and the metabolites 1-(3,4-dichlorophenyl) urea (DCU), 3,4-dichloroaniline (DCA) and 1-(3,4-dichlorophenyl)-3-methoxyurea (DCXU) for androgen receptor (AR) activities and effects on steroidogenesis. Generally, linuron and the metabolites showed qualitatively similar antiandrogenic profiles, but potencies varied. All compounds were AR antagonists, with linuron showing highest potency (IC50 of 2.8 µM). The overall picture of effects on steroidogenesis showed that linuron and metabolites increased the levels of estrogens and corticosteroids, whereas the synthesis of androgens was inhibited. The metabolite DCU was by far the most potent inhibitor of testosterone synthesis (IC50 of 6.7 µM compared to IC50 of 51.1 µM for linuron). We suggest that it is likely that the metabolites contribute to the antiandrogenic effects of linuron in vivo, especially by inhibiting testosterone synthesis.

Exposure to the pesticides linuron, dimethomorph and imazalil alters steroid hormone profiles and gene expression in developing rat ovaries.

Inhibition of androgen signaling during critical stages of ovary development can disrupt folliculogenesis with potential consequences for reproductive function later in life. Many environmental chemicals can inhibit the androgen signaling pathway, which raises the question if developmental exposure to anti-androgenic chemicals can negatively impact female fertility. Here, we report on altered reproductive hormone profiles in prepubertal female rats following developmental exposure to three pesticides with anti-androgenic potential: linuron (25 and 50 mg/kg bw/d), dimethomorph (60 and 180 mg/kg bw/d) and imazalil (8 and 24 mg/kg bw/d). Dams were orally exposed from gestational day 7 (dimethomorph and imazalil) or 13 (linuron) until birth, then until end of dosing at early postnatal life. Linuron and dimethomorph induced dose-related reductions to plasma corticosterone levels, whereas imazalil mainly suppressed gonadotropin levels. In the ovaries, expression levels of target genes were affected by linuron and dimethomorph, suggesting impaired follicle growth. Based on our results, we propose that anti-androgenic chemicals can negatively impact female reproductive development. This highlights a need to integrate data from all levels of the hypothalamic-pituitary-gonadal axis, as well as the hypothalamic-pituitary-adrenal axis, when investigating the potential impact of endocrine disruptors on female reproductive development and function.

Changes in soil microbial community and activity caused by application of dimethachlor and linuron.

Soil microorganisms and their activities are essential for maintaining soil health and fertility. Microorganisms can be negatively affected by application of herbicides. Although effects of herbicides on microorganisms are widely studied, there is a lack of information for chloroacetamide herbicide dimethachlor. Thus, dimethachlor and well known linuron were applied to silty-loam luvisol and their effects on microorganisms were evaluated during112 days long laboratory assay. Dimethachlor and linuron were applied in doses 1.0 kg ha-1 and 0.8 kg ha-1 corresponding to 3.33 mg kg-1 and 2.66 mg kg-1 respectively. Also 100-fold doses were used for magnification of impacts. Linuron in 100-fold dose caused minor increase of respiration, temporal increase of soil microbial biomass, decrease of soil dehydrogenase activity, and altered microbial community. Dimethachlor in 100-fold dose significantly increased respiration; microbial biomass and decreased soil enzymatic activities. Microbial composition changed significantly, Proteobacteria abundance, particularly Pseudomonas and Achromobacter genera increased from 7 to 28th day. In-silico prediction of microbial gene expression by PICRUSt2 software revealed increased expression of genes related to xenobiotic degradation pathways. Evaluated characteristics of microbial community and activity were not affected by herbicides in recommended doses and the responsible use of both herbicides will not harm soil microbial community.

An Experimental Approach to Study the Effects of Realistic Environmental Mixture of Linuron and Propamocarb on Zebrafish Synaptogenesis.

The reasons behind the extensive use of pesticides include the need to destroy vector organisms and promote agricultural production in order to sustain population growth. Exposure to pesticides is principally occupational, even if their persistence in soil, surface water and food brings the risk closer to the general population, hence the demand for risk assessment, since these compounds exist not only as individual chemicals but also in form of mixtures. In light of this, zebrafish represents a suitable model for the evaluation of toxicological effects. Here, zebrafish embryos were exposed for 96 h post fertilization (hpf) to sublethal concentrations (350 µg/L) of linuron and propamocarb, used separately and then combined in a single solution. We investigated the effects on morphological traits and the expression of genes known to be implicated in synaptogenesis (neurexin1a and neuroligin3b). We observed alterations in some phenotypic parameters, such as head width and interocular distance, that showed a significant reduction (p < 0.05) for the mixture treatment. After individual exposure, the analysis of gene expression showed an imbalance at the synaptic level, which was partially recovered by the simultaneous administration of linuron and propamocarb. This preliminary study demonstrates that the combined substances were responsible for some unpredictable effects, diverging from the effect observed after single exposure. Thus, it is clear that risk assessment should be performed not only on single pesticides but also on their mixtures, the toxicological dynamics of which can be totally unpredictable.

Assessment of oxidative DNA damage, oxidative stress responses and histopathological alterations in gill and liver tissues of Oncorhynchus mykiss treated with linuron.

We investigated changes in 8-hydroxy-2-deoxyguanosine (8-OHdG) activity which is a product of oxidative DNA damage, histopathological changes and antioxidant responses in liver and gill tissues of rainbow trout, following a 21-day exposure to three different concentrations of linuron (30 µg/L, 120 µg/L and 240 µg/L). Our results indicated that linuron concentrations caused an increase in LPO levels of liver and gill tissues (p < 0.05). While linuron induced both increases and decreases in GSH levels and SOD activity, CAT activity was decreased by all concentrations of linuron (p < 0.05). The immunopositivity of 8-OHdG was detected in the hepatocytes of liver and in the epithelial and chloride cells of the secondary lamellae of the gill tissues. Our results suggested that linuron could cause oxidative DNA damage by causing an increase in 8-OHdG activity in tissues, and it induces histopathological damage and alterations in the antioxidant parameters of the tissues.

Evaluation of dietary dose administration as an alternative to oral gavage in the rodent uterotrophic and Hershberger assays.

The rodent uterotrophic and Hershberger assays evaluate potential estrogenic and (anti)-androgenic effects, respectively. Both US EPA and OECD guidelines specify that test substance is administered daily either by subcutaneous injection or oral gavage. However, dietary administration is a relevant exposure route for agrochemical regulatory toxicology studies due to potential human intake via crop residues. In this study, equivalent doses of positive control chemicals administered via dietary and gavage routes of administration were compared in the uterotrophic (17α-ethinyl estradiol) and Hershberger (flutamide, linuron, dichloro-2,2-bis(4-chlorophenyl) ethane; 4,4'-DDE) assays in ovariectomized and castrated rats, respectively. For all positive control chemicals tested, statistically significant changes in organ weights and decreases in food consumption were observed by both routes of test substance administration. Decreased body weight gain observed for dietary linuron and 4,4'-DDE indicated that the maximum tolerated dose was exceeded. Hershberger dietary administration resulted in a similar blood exposure (AUC24) for each positive control chemical when compared to gavage. Overall, the correlation in organ weight changes for both the uterotrophic and Hershberger assays suggest that dietary administration is an acceptable route of exposure with similar sensitivity to oral gavage dosing for evaluation of the endocrine potential of a test substance and represents a more appropriate route of test substance administration for most environmental exposure scenarios.

Quantitative in Vitro to in Vivo Extrapolation (QIVIVE) for Predicting Reduced Anogenital Distance Produced by Anti-Androgenic Pesticides in a Rodent Model for Male Reproductive Disorders.

BACKGROUND: Many pesticides can antagonize the androgen receptor (AR) or inhibit androgen synthesis in vitro but their potential to cause reproductive toxicity related to disruption of androgen action during fetal life is difficult to predict. Currently no approaches for using in vitro data to anticipate such in vivo effects exist. Prioritization schemes that limit unnecessary in vivo testing are urgently needed. OBJECTIVES: The aim was to develop a quantitative in vitro to in vivo extrapolation (QIVIVE) approach for predicting in vivo anti-androgenicity arising from gestational exposures and manifesting as a shortened anogenital distance (AGD) in male rats. METHODS: We built a physiologically based pharmacokinetic (PBK) model to simulate concentrations of chemicals in the fetus resulting from maternal dosing. The predicted fetal levels were compared with analytically determined concentrations, and these were judged against in vitro active concentrations for AR antagonism and androgen synthesis suppression. RESULTS: We first evaluated our model by using in vitro and in vivo anti-androgenic data for procymidone, vinclozolin, and linuron. Our PBK model described the measured fetal concentrations of parent compounds and metabolites quite accurately (within a factor of five). We applied the model to nine current-use pesticides, all with in vitro evidence for anti-androgenicity but missing in vivo data. Seven pesticides (fludioxonil, cyprodinil, dimethomorph, imazalil, quinoxyfen, fenhexamid, o-phenylphenol) were predicted to produce a shortened AGD in male pups, whereas two (λ-cyhalothrin, pyrimethanil) were anticipated to be inactive. We tested these expectations for fludioxonil, cyprodinil, and dimethomorph and observed shortened AGD in male pups after gestational exposure. The measured fetal concentrations agreed well with PBK-modeled predictions. DISCUSSION: Our QIVIVE model newly identified fludioxonil, cyprodinil, and dimethomorph as in vivo anti-androgens. With the examples investigated, our approach shows great promise for predicting in vivo anti-androgenicity (i.e., AGD shortening) for chemicals with in vitro activity and for minimizing unnecessary in vivo testing. https://doi.org/10.1289/EHP6774.

Maternal exposure to mixtures of dienestrol, linuron and flutamide. Part II: Endocrine-related gene expression assessment on male offspring rat testes.

Exposure to endocrine-disrupting compounds (EDCs) during pregnancy and early development can lead to adverse developmental outcomes in offspring. One of the endpoints of concern is feminization. The present study aimed to investigate for any possible correlations with endocrine sensitive parameters in the testes of male rat offspring following dam exposure to three EDCs by assessing the expression of endocrine-related genes. Dienestrol (DIES) [0.37-6.25 µg/kg bw/day], linuron (LIN) [1.5-50 mg/kg bw/day], flutamide (FLU) [3.5-50 mg/kg bw/day] as well as their binary mixtures were administered to sexually mature female rats from gestation day (GD) 6 until postnatal day (PND) 21. Gene expression analysis of Star, Cyp11a1, Cyp17a1, Hsd3b2, Pgr and Insl3 was performed by RT-qPCR. Administration of the anti-androgen FLU alone significantly upregulated Cyp11a1 and Cyp17a1 gene expression while administration of LIN and DIES alone did not alter significantly gene expression. The effects of the binary mixtures on gene expression were not as marked as those seen after single compound administrations. Deregulation of Cyp17a1 in rat pup testis, following administration of FLU alone or in mixtures to dams, was significantly correlated with the observed feminization endpoints in male pups.

Maternal exposure to mixtures of dienestrol, linuron and flutamide. Part I: Feminization effects on male rat offspring.

Exposure to endocrine-disrupting compounds (EDCs) during pregnancy can result in negative health effects in later generations, including sex changes and feminization. The present study assessed the feminization effects on male offspring rats of three EDCs: Dienestrol (DIES), Linuron (LIN), and Flutamide (FLU). Sexually mature female rats were exposed from gestation day (GD) 6 until postnatal day (PND) 21 to: 0.37, 0.75, 1.5, 3.12 or 6.25 µg/kg/day of DIES, 1.5, 3, 6, 12.5, 25 or 50 mg/kg/day of LIN, 3.5, 6.7, 12.5, 25 or 50 mg/kg/day of FLU, and the following mixtures: FLU + DIES (mg/kg/day+µg/kg/day), 3.5 + 0.37, or 3.5 + 3, 25 + 0.37, or 25 + 3; FLU + LIN (mg/kg/day + mg/kg/day), 3.5 + 12.5, or 25 + 12.5; and DIES + LIN (µg/kg/day + mg/kg/day), 0.37 + 12.5, or 3 + 12.5. Anogenital distance (AGD), nipple retention (NR) and cryptorchidism were evaluated. FLU produced a decrease of AGD, an increase of NR, and an increase of cryptorchidism at the highest dose. None of these three endpoints were significantly affected by LIN or DIES treatments alone. Combinations of FLU + LIN and FLU + DIES increased NR, and decreased AGD, while DIES + LIN did not produce any effects in male pups. Results show that FLU is able to induce feminization in male pups, while binary combinations of LIN and DIES did not modify the effects produced by FLU.

Exposure to herbicide linuron results in alterations in hematological profile and stress biomarkers of common carp (Cyprinus carpio).

Phenylurea herbicides such as linuron are commonly applied in agriculture. Common carp juveniles were subjected to 31.5 µg/L of linuron for 14 days, and then to 30 days of purification. Peripheral blood was sampled after 1, 3, 7 and 14 days of exposure and 7, 14 and 30 days of purification and hematological parameters were evaluated: erythrocyte (RBCc) and leukocyte (WBCc) counts, hematocrit (Ht), hemoglobin concentration (Hb), mean cell volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and differential leukocyte count. For evaluation of cortisol and catecholamine concentrations blood was sampled after 3, 6 and 12 h, after 1, 3 and 14 days of exposure, and after 30 days of purification. Linuron caused mainly transient increase in RBCc, Ht and MCV values and increase in WBCc and percentage of juvenile neutrophils. The herbicide caused persistant increase of cortisol and catecholamine concentrations. The results indicate that exposure to low concentration of linuron induced a stress response in common carp.

Expression of the human gene CYP1A2 enhances tolerance and detoxification of the phenylurea herbicide linuron in Arabidopsis thaliana plants and Escherichia coli.

The phenylurea herbicide, linuron (LIN), is used to control various types of weeds. Despite its efficient role in controlling weeds, it presents a persistent problem to the environment. In the current study, phytoremediation properties of transgenic CYP1A2 Arabidopsis thaliana plants to LIN were assessed. CYP1A2 gene was firstly cloned and expressed in bacteria before proceeding to plants. In presence of LIN, The growth of CYP1A2 expressing bacteria was superior compared to control bacteria transformed with the empty bacterial expression vector pET22b(+). No clear morphological changes were detected on CYP1A2 transgenic plants. However, significant resistance to LIN herbicide application either via spraying the foliar parts of the plant or via supplementation of the herbicide in the growth medium was observed for CYP1A2 transformants. Plant growth assays under LIN stress provide strong evidence for the enhanced capacity of transgenic lines to grow and to tolerate high concentrations of LIN compared to control plants. HPLC analyses showed that detoxification of LIN by bacterial extracts and/or transgenic plant leaves is improved as compared to the corresponding controls. Our data indicate that over expression of the human CYP1A2 gene increases the phytoremediation capacity and tolerance of Arabidopsis thaliana plants to the phenylurea herbicide linuron.

Maternal linuron exposure alters testicular development in male offspring rats at the whole genome level.

Linuron is a widely used herbicide; its toxicity on the male reproductive system has been recognized. The current study was designed to explore the molecular mechanism underlying linuron-induced reproductive toxicity. Pregnant rats received daily oral gavage of linuron at the dose of 120mg/kg/d from gestation day (GD)12 to GD17. Tissues from male offspring rats were collected for pathological examination and microarray gene expression profiling. Changes in gene expression were further verified by quantitative real-time RT-PCR. Data showed that linuron-exposed offspring rats had a decreased sperm count (88% of controls) and disrupted acrosome formation. There were evident damages in seminiferous tubules and abnormal morphology in mesenchymal cells in samples from linuron-exposed animals. Microarray analysis indicated that the expressions of testosterone synthesis-associated genes, i.e., Star, P450scc, 3ß-Hsd, Abp, Cox7a2, Pcna, p450c17and17ß-Hsd were significantly altered by linuron exposure, along with other genes involving in cell proliferation and apoptosis, such as c-myc, S6K, Apaf1, and TSC1. These data indicate that linuron upon entering male offspring body can directly or indirectly interact with the androgen production and function; linuron-induced alteration in genes encoding testosterone synthesis is likely a major factor in linuron-induced male reproductive toxicity.

Endocrine and physiological effects of linuron and S-metolachlor in zebrafish developing embryos.

Evaluation of the effects of linuron and S-metolachlor on apical, biochemical and transcriptional endpoints in zebrafish (Danio rerio) early life stages was the main purpose of this work. Embryos were exposed for 96h to a range of concentrations of each herbicide to determine lethal and sub-lethal effects on apical (e.g. malformations, hatching) and biochemical parameters (cholinesterase, ChE; catalase, CAT; glutathione S-transferase, GST; lipid peroxidation, LPO and lactate dehydrogenase, LDH). To evaluate endocrine disruption effects, embryos were exposed during 96h to 0.88mg/L linuron and 9.66mg/L S-metolachlor, isolated or in binary mixture. Expression of a suite of genes involved in HPT, HPG and HPA-axis was then assessed. Highest concentration of linuron (5.0mg/L) decreased hatching rate to 5% and 70.0mg/L S-metolachlor completely inhibited hatching, about 100%. Both herbicides impaired development by inducing several malformations (100% in 5.0mg/L linuron and 70.0mg/L S-metolachlor). Linuron only affected GST and CAT at concentrations of 0.25 and 0.0025mg/L, respectively. S-metolachlor induced GST (to 256%), inhibited ChE (to 61%) and LDH (to 60%) and reduced LPO levels (to 63%). Linuron isolated treatment seems to have an estrogenic mode of action due to the observed induction of vtg1. Exposure to S-metolachlor seems to interfere with steroidogenesis and with HPT and HPA-axis, since it has inhibited cyp19a2, TSHß and CRH gene expression. In addition to vtg1 induction and CRH inhibition, herbicide combination also induced sox9b that has a role in regulation of sexual development in zebrafish. This study pointed out adverse effects of linuron and S-metolachlor, namely impairment of neurotransmission and energy production, induction of steroidogenesis, and interference with HPT and HPA-axis. These results contributed to elucidate modes of action of linuron and S-metolachlor in zebrafish embryo model. Furthermore, gene expression patterns obtained are indicative of endocrine disruption action of these herbicides.

Biological responses to phenylurea herbicides in fish and amphibians: New directions for characterizing mechanisms of toxicity.

Urea-based herbicides are applied in agriculture to control broadleaf and grassy weeds, acting to either inhibit photosynthesis at photosystem II (phenylureas) or to inhibit acetolactate synthase acetohydroxyacid synthase (sulfonylureas). While there are different chemical formulas for urea-based herbicides, the phenylureas are a widely used class in North America and have been detected in aquatic environments due to agricultural run-off. Here, we summarize the current state of the literature, synthesizing data on phenylureas and their biological effects in two non-target animals, fish and amphibians, with a primary focus on diuron and linuron. In fish, although the acutely lethal effects of diuron in early life stages appear to be >1mg/L, recent studies measuring sub-lethal behavioural and developmental endpoints suggest that diuron causes adverse effects at lower concentrations (i.e. <0.1mg/L). Considerably less toxicity data exist for amphibians, and this is a knowledge gap in the literature. In terms of sub-lethal effects and mode of action (MOA), linuron is well documented to have anti-androgenic effects in vertebrates, including fish. However, there are other MOAs that are not adequately assessed in toxicology studies. In order to identify additional potential MOAs, we conducted in silico analyses for linuron and diuron that were based upon transcriptome studies and chemical structure-function relationships (i.e. ToxCast™, Prediction of Activity Spectra of Substances). Based upon these analyses, we suggest that steroid biosynthesis, cholesterol metabolism and pregnane X receptor activation are common targets, and offer some new endpoints for future investigations of phenylurea herbicides in non-target animals.

Reproductive toxicity of linuron following gestational exposure in rats and underlying mechanisms.

Linuron is a widely used herbicide in agriculture; its endocrine disruptive toxicity has recently received public attention. This study was designed to examine the developmental toxicity of linuron on the reproductive system of male offspring following maternal exposure. Mother rats received oral gavages of linuron, once daily, at the dose of 0, 50, 100, 150 or 200mg/kg, from gestational day (GD)13 to GD18; gonadal organs from GD20 fetuses were examined. Data indicated that exposed male offspring had a significantly shortened anogenital distance. Pathological examination further revealed a lack of fusion in the urogenital fold in treated fetuses, the damaged seminiferous tubules, and the injured Leydig cell ultrastructure. Analysis of serum testosterone concentrations at postnatal day (PND)2 showed a significant dose-related reduction (about 33.7-58.75%, r=-0.838, p<0.05) as compared to controls. Immunohistochemical results demonstrated a significantly reduced expression of enzymes pertinent to the testosterone production including P450scc, 3ß-HSD, and PCNA in Leydig cells (p<0.05). qPCR studies confirmed decreased levels of mRNAs encoding P450scc, 3ß-HSD and PCNA (p<0.05). Taken together, these data suggest that maternal exposure to linuron hampers the male gonadal organ development; this appears to be due to linuron's direct action on the production of testosterone in fetal and postnatal offspring.

Evaluation of 8-hydroxy-2-deoxyguanosine and NFkB activation, oxidative stress response, acetylcholinesterase activity, and histopathological changes in rainbow trout brain exposed to linuron.

Linuron is a widely used herbicide to control grasses and annual broad leaf weeds. It is known that linuron has toxic effects on different organisms. However, the toxic effects of linuron on aquatic organisms, especially fish, is completely unknown. Thus, we aimed to investigate changes in 8-hydroxy-2-deoxyguanosine (8-OHdG) and nuclear factor kappa B (NFkB) activity, histopathological changes, antioxidant responses and acetylcholinesterase (AChE) activity in rainbow trout brain after exposure to linuron. Fish were exposed to 30µg/L, 120µg/L and 240µg/L concentrations of linuron for twenty-one days. Brain tissues were taken from fish for 8-OHdG and NFkB activity, histopathological examination and determination of superoxide dismutase (SOD), catalase (CAT) enzyme activity, lipid peroxidation (LPO), and reduced glutathione (GSH) levels. Our data indicated that high linuron concentrations caused a decrease in GSH levels, SOD and CAT activities in brain tissues (p<0.05). LPO levels were significantly increased by 240µg/L linuron. All concentrations caused a significant inhibition in brain AChE enzyme activity (p<0.05). Immunopositivity was detected for 8-OHdG and NFkB, and linuron exposure caused histopathological damage to the brain tissues. The results of this study can provide useful information for understanding of linuron-induced toxicity.

Effects of anti-androgens cyproterone acetate, linuron, vinclozolin, and p,p'-DDE on the reproductive organs of the copepod Acartia tonsa.

The study was performed to detect the effects of anti-androgenic compounds on the reproduction. In this paper alterations observed in the marine calanoid copepod Acartia tonsa exposed to environmental concentrations of cyproterone acetate (CPA), linuron (LIN), vinclozolin (VIN), and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE) for 21 days covering a full life cycle are described. Histological alterations were studied with a focus on reproductive organs, gonad and accessory sexual glands. Exposure to ≥1.2 µg L(-1) CPA caused degeneration of spermatocytes and deformation of the spermatophore in males. In a single male exposed to 33 µg L(-1) CPA, an ovotestis was observed. In CPA exposed females, enhancement of oogenesis, increase in apoptosis and a decrease in proliferation occurred. Exposure of males to ≥12 µg L(-1) LIN caused degenerative effects in spermatogonia, spermatocytes and spermatids, and at 4.7 µg L(-1) LIN, the spermatophore wall displayed an irregular formation. In LIN exposed females, no such structural alterations were found; however, the proliferation index was reduced at 29 µg L(-1) LIN. At an exposure concentration of ≥100 µg L(-1) VIN, distinct areas in male gonad were stimulated, whereas others displayed a disturbed spermatogenesis and a deformed spermatophore wall. In VIN exposed female A. tonsa, no effects were observed. Male A. tonsa exposed to p,p'-DDE displayed an impairment of spermatogenesis in all stages with increased degrees of apoptosis. In p,p'-DDE-exposed females, a statistical significant increase of the proliferation index and an intensification of oogenesis were observed at 0.0088 µg L(-1).

Mutagenic properties of linuron and chlorbromuron evaluated by means of cytogenetic biomarkers in mammalian cell lines.

Agricultural practices are usually supported by several chemical substances, such as herbicides. Linuron and chlorbromuron are phenylurea herbicides largely used to protect crops from weeds, blocking photosynthesis by inhibition of the photosystem II complex. The former, also commercially known as lorox or afalon, is selectively used to protect bean and French bean plants, fennels, and celeriacs; the second, commercially known as maloran, is selectively used for carrots, peas, potatoes, soy sprouts, and sunflowers. Considering the widespread use of herbicides and, more generally, pesticides, it is important to clarify their involvement on human health, one of them concerning the possible direct or indirect effect on the genome of exposed populations. Here, we show that these herbicides are endowed by mutagenic properties, as demonstrated by an increased number of chromosomal aberrations (CAs) in two exposed Chinese hamster cell lines derived from ovary and epithelial liver, respectively. This was also confirmed by sister chromatid exchange (SCE) and micronucleus (MN) assays. Our present and previously obtained data clearly indicate that phenylurea herbicides must be used with great caution, especially for agricultural workers who use large amounts of herbicides during their work, and particular attention should be given to residues of these herbicides and their involvement in environmental pollution.

The herbicide linuron inhibits cholesterol biosynthesis and induces cellular stress responses in brown trout.

The herbicide linuron is used worldwide, and has been detected in surface waters as well as in food and drinking water. Toxicological studies have reported that linuron acts as an antiandrogen in vitro and in vivo and disrupts mammalian male reproductive function. However, global mechanisms of linuron toxicity are poorly documented. We used RNA-seq to characterize the hepatic transcriptional response of mature male brown trout exposed for 4 days to 1.7, 15.3, and 225.9 µg/L linuron. We identified a striking decrease in the expression of transcripts encoding the majority of enzymes forming the cholesterol biosynthesis pathway. We also measured a very significant decrease in total hepatic cholesterol in fish exposed to 225.9 µg/L linuron and a negative correlation between total cholesterol and linuron treatment concentration. We hypothesize that inhibition of cholesterol biosynthesis may result from the disruption of androgen signaling by linuron. Additionally, there was increased expression of a number of transcripts involved in cellular stress responses, including cyp1a (up to 560-fold), molecular chaperones, and antioxidant enzymes. We found some evidence of similar patterns of transcriptional change in fish exposed to an environmentally relevant concentration of linuron, and further research should investigate the potential for adverse effects to occur following chronic environmental exposure.

Assessing single and joint toxicity of three phenylurea herbicides using Lemna minor and Vibrio fischeri bioassays.

Single and joint toxicity of three substituted urea herbicides, namely monolinuron [3-(4-chlorophenyl)-1-methoxy-1-methylurea], linuron [3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea] and diuron [1-(3,4 dichlorophenyl)-3,3 dimethyl urea], were studied. The duckweed Lemna minor and the luminescent bacterium Vibrio fischeri were used for the toxicity assessment and they were exposed to various concentrations of the herbicides, individually and in binary mixtures. The exposure time was 7d for the duckweed and 30 min for the bacterium. Estimation of EC50 values was performed by frond counting and reduction in light output for Lemna minor and Vibrio fischeri, respectively. Lemna minor was found to be much more sensitive than Vibrio fischeri to target compounds. The toxicity of the three herbicides applied solely was estimated to be in decreasing order: diuron (EC50=28.3 µg L(-1))≈linuron (EC50=30.5 µg L(-1))>monolinuron (EC50=300 µg L(-1)) for the duckweed and linuron (EC50=8.2 mg L(-1))>diuron (EC50=9.2 mg L(-1))>monolinuron (EC50=11.2 mg L(-1)) for the bacterium. Based on the environmental concentrations reported in the literature and EC50 values obtained from Lemna minor experiments, Risk Quotients (RQ) much higher than 1 were calculated for diuron and linuron. In Lemna minor experiments, combination of target compounds resulted to additive effects due to their same mode of phenylurea action on photosynthetic organisms. Regarding Vibrio fischeri, synergistic, additive and antagonistic effects were observed, which varied according to the concentrations of target compounds.