Herbicides widely used in the world: an investigation of toxic effects on Caenorhabditis elegans.

Dicamba, paraquat, picloram, clopyralid and linuron are herbicides widely used in agriculture. The aim of the present study is to evaluate the toxicity effects of the herbicides used on survival, fertility and length of Caenorhabditis elegans. Kaplan-Meier Survival Analysis method was used to identify the toxicity effect of herbicides on survival, and ANOVA and Post Hoc tests were used to determine the toxicity effects on fertility and length. In the study, C. elegans was exposed to 5 different concentrations (62.5, 125, 250, 500, 1000 µM) of each herbicide. When the results were evaluated, it was observed that survival (life span) and length (physical growth) were more affected, respectively, by paraquat, dicamba, linuron, picloram and clopyralid herbicides, fertility (egg productivity) were more affected, respectively, by paraquat, linuron, dicamba, picloram and clopyralid herbicides. As a result, it was determined that increasing the dose amounts of herbicides caused many toxic reactions on C. elegans, affecting survival, egg productivity and length.

Interactive effects of high temperature and pesticide exposure on oxidative status, apoptosis, and renin expression in kidney of goldfish: Molecular and cellular mechanisms of widespread kidney damage and renin attenuation.

One of many noteworthy consequences of increasing societal reliance on pesticides is their predominance in aquatic environments. These pernicious chemicals interact with high temperatures from global climate change, heat waves, and natural variations to create unstable environments that negatively impact organisms' health. To understand these conditions, we examined the dose-dependent effects of environmentally relevant pesticide mixtures (metolachlor, linuron, isoproturon, tebuconazole, aclonifen, atrazine, pendimethalin, and azinphos-methyl) combined with elevated temperatures (22 control vs. 32°C for 4-week exposure) on renin, dinitrophenyl protein (DNP, an indicator of reactive oxygen species, ROS), 3-nitrotyrosine protein (NTP, an indicator of reactive nitrogen species, RNS), superoxidase dismutase (SOD, an antioxidant), and catalase (CAT, an antioxidant) expressions in the kidneys of goldfish (Carassius auratus). Histopathological analysis showed widespread damage to kidney tissues in high temperature and pesticide co-exposure groups, including rupture of the epithelial layer, hemorrhaging, and degeneration of tubular epithelium. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemical analyses demonstrated significant declines in renin receptor-like mRNA and protein expressions in kidney tissues under combined exposure to high temperature and pesticides compared with controls; conversely, expression of DNP, NTP, SOD, and CAT increased in kidney tissues under the same conditions. Apoptotic cells were also increased in co-exposure groups as assessed by in situ terminal deoxynucleotidyl transferase dUTP nick labeling (TUNEL) assay. The enhanced apoptosis in kidneys of heat and pesticides co-exposed fish was associated with increased caspase-3 (a protease enzyme) mRNA levels. Our results demonstrated that high temperature and pesticides induced oxidative/nitrative stress (i.e., ROS/RNS), damaged tissues, increased cellular apoptosis, and suppressed renin expression in kidneys of goldfish.

Sub-lethal toxicity assessment of the phenylurea herbicide linuron in developing zebrafish (Danio rerio) embryo/larvae.

Due to run-off and rain events, agrochemicals can enter water catchments, exerting endocrine disruption effects and toxicity to aquatic organisms. Linuron is a phenylurea herbicide used to control a wide variety of vegetative weeds in agriculture in addition to residential applications. However, there are few studies that quantify its toxicity to early developmental stages of fish. The objectives of this study were to assess the acute toxicity of linuron to zebrafish embryos/larvae by measuring mortality, morphological deformities, oxidative respiration, gene expression, and locomotor activity via the Visual Motor Response test. Zebrafish embryos at ~6-h post-fertilization (hpf) were exposed to either embryo rearing medium (ERM), or one dose of 0.625, 1.25, 2.5, 5, and 10 µM linuron for up to 7 days post-fertilization (dpf) depending on the assay. Zebrafish larvae exposed to linuron displayed pericardial edema, yolk sac edema, and spinal curvature. Oxidative respiration assessments in embryos using the Agilent XFe24 Flux Analyzer revealed that linuron decreased mean basal respiration and oligomycin-induced ATP-linked respiration in 30 hpf embryos at 20 µM after a 24-hour exposure. In 7 dpf larvae, transcript abundance was determined for 6 transcripts that have a role in oxidative respiration (atp06, cox1, cox4-1, cox5a1, cytb, and nd1); the relative abundance of these transcripts was not altered with linuron treatment. A Visual Motor Response test was conducted on 7 dpf larvae to determine whether linuron (0.625 to 5 µM) impaired locomotor activity. Larval activity in the dark period decreased in a dose dependent manner and there were indications of hypoactivity as low as 1.25 µM. Transcript abundance was thus determined for tyrosine hydroxylase (th1) and glutamic acid decarboxylase 67 (gad1b), two rate limiting enzymes that control the production of dopamine and gamma-aminobutyric acid respectively. The mRNA levels of gad1b (p = 0.019) were reduced with increasing concentrations of linuron while th1 (p = 0.056) showed a similar decreasing trend, suggesting that neurotransmitter biosynthesis may be altered with exposure to linuron. This study improves knowledge related to the toxicity mechanisms for linuron and is the first to demonstrate that this anti-androgenic chemical impairs oxidative respiration and exerts neurotoxic effects associated with neurotransmitter biosynthesis during early development. These data are significant for environmental risk assessment of agrochemicals.

Removal of batch effects using stratified subsampling of metabolomic data for in vitro endocrine disruptors screening.

The human adrenal cell line H295R constitutes a well-established model to evaluate potential alterations of steroidogenic pathways as a result of chemical exposure. However, to date most assays are based on the targeted investigation of a limited number of steroid hormones, thus preventing in-depth mechanistic interpretation with respect to steroidogenesis. In that context, analytical strategies coupling liquid chromatography and high-resolution mass spectrometry (LC-HRMS) have been reported as promising methods for an extended monitoring of steroid metabolites. However, unwanted sources of variability occurring during the acquisition process, including batch effects, may prevent relevant biochemical information to be properly highlighted. Dedicated data mining strategies are therefore needed to overcome these limitations, and extract relevant extended steroidomic profiles. The present study combines an untargeted LC-HRMS acquisition strategy with automated steroid metabolite annotation based on accurate mass and isotopic patterns, and a chemometric tool allowing the different sources of variability to be decomposed based on experimental design. This workflow was applied to the extended monitoring of steroidogenic dysregulations due to endocrine disrupting chemicals (EDCs) exposure in H295R cell cultures. A series of six chemicals, including acetyl tributylcitrate, octyl methoxycinnamate, torcetrapib, forskolin, linuron, and octocrylene, and dimethylsulfoxide as solvent control, were investigated through the simultaneous monitoring of 130 potential steroid metabolites, repeating the whole experiment independently three times. A stratified subsampling strategy was carried out to remove efficiently systematic batch variations and highlight subgroups of chemicals with similar steroid patterns. The proposed approach was reported as a potent screening strategy, as it allowed specific alterations of the steroid hormone biosynthesis and metabolism related to distinct mechanisms of action to be distinguished.

Effects of the herbicides linuron and S-metolachlor on Perez's frog embryos.

Presence of pesticides in the environment and their possible effects on aquatic organisms are of great concern worldwide. The extensive use of herbicides in agricultural areas are one of the factors contributing to the known decline of amphibian populations. Thus, as non-target species, amphibians can be exposed in early life stages to herbicides in aquatic systems. In this context, this study aims to evaluate effects of increasing concentrations of two maize herbicides, linuron and S-metolachlor on embryos of the Perez' frog (Pelophylax perezi) during 192 h. Apical endpoints were determined for each herbicide: mortality, hatching rate, malformations and length. Frog embryos presented a LC50 of 21 mg/l linuron and 37.5 mg/l S-metolachlor. Furthermore, sub-lethal concentrations of both herbicides affected normal embryonic development, delaying hatching, decreasing larvae length and causing several malformations. Length of larvae decreased with increasing concentrations of each herbicide, even at the lower concentrations tested. Malformations observed in larvae exposed to both herbicides were oedemas, spinal curvature and deformation, blistering and microphtalmia. Overall, these results highlight the need to assess adverse effects of xenobiotics to early life stages of amphibians regarding beside mortality the embryonic development, which could result in impairments at later stages. However, to unravel mechanisms involved in toxicity of these herbicides further studies regarding lower levels of biological organisation such as biochemical and genomic level should be performed.

The inhibition of myometrial contractions by chlorinated herbicides (atrazine and linuron), and their disruptive effect on the secretory functions of uterine and ovarian cells in cow, in vitro.

The effect of atrazine and linuron, the popular and widely used chlorinated herbicides, on both myometrial contractions and secretory functions of bovine uterus and ovaries in vitro, was investigated. The pesticides inhibited (P<0.05) the basal and oxytocin (OT)-stimulated myometrial strips contractions, as well as the effect of OT on secretion of prostaglandins (PGs: PGF2α and PGE2) from endometrium. But only linuron inhibits the effect of OT on myometrial contractions. Neither of herbicides affected PGs secretion from myometrium and PGF2α from endometrium. Only the lowest dose of both tested compounds decreased PGE2 secretion from endometrium. The pesticides increased (P<0.05) the OT secretion from granulosa. However, only linuron stimulated (P<0.05) the OT secretion from the luteal cells, and it increased (P<0.05) the expression of mRNA for the OT precursor. Both compounds stimulated (P<0.05) the secretion of testosterone and atrazine increased (P<0.05) also the secretion of estradiol from the granulosa cells. While atrazine and linuron reduced (P<0.05) the progesterone secretion from the luteal cells. The data show that atrazine and linuron altered the secretory functions of ovarian cells and inhibited the myometrial contractions in vitro.

Using short-term bioassays to evaluate the endocrine disrupting capacity of the pesticides linuron and fenoxycarb.

Several short-term whole-organism bioassays based on transgenic aquatic models are now under validation by the OECD (Organization for Economic Co-operation and Development) to become standardized test guidelines for the evaluation of the endocrine activity of substances. Evaluation of the endocrine disrupting capacity of pesticides will be a domain of applicability of these future reference tests. The herbicide linuron and the insecticide fenoxycarb are two chemicals commonly used in agricultural practices. While numerous studies indicate that linuron is likely to be an endocrine disruptor, there is little information available on the effect of fenoxycarb on vertebrate endocrine systems. Using whole-organism bioassays based on transgenic Xenopus laevis tadpoles and medaka fry we assessed the potential of fenoxycarb and linuron to disrupt thyroid, androgen and estrogen signaling. In addition we used in silico approach to simulate the affinity of these two pesticides to human hormone receptors. Linuron elicited thyroid hormone-like activity in tadpoles at all concentrations tested and, showed an anti-estrogenic activity in medaka at concentrations 2.5mg/L and higher. Our experiments suggest that, in addition to its previously established anti-androgenic action, linuron exhibits thyroid hormone-like responses, as well as acting at the estrogen receptor level to inhibit estrogen signaling. Fenoxycarb on the other hand, did not cause any changes in thyroid, androgen or estrogen signaling at the concentrations tested.

Evidence of in vitro metabolic interaction effects of a chlorfenvinphos, ethion and linuron mixture on human hepatic detoxification rates.

General population exposure to pesticides mainly occurs via food and water consumption. However, their risk assessment for regulatory purposes does not currently consider the actual co-exposure to multiple substances. To address this concern, relevant experimental studies are needed to fill the lack of data concerning effects of mixture on human health. For the first time, the present work evaluated on human microsomes and liver cells the combined metabolic effects of, chlorfenvinphos, ethion and linuron, three pesticides usually found in vegetables of the European Union. Concentrations of these substances were measured during combined incubation experiments, thanks to a new analytical methodology previously developed. The collected data allowed for calculation and comparison of the intrinsic hepatic clearance of each pesticide from different combinations. Finally, the results showed clear inhibitory effects, depending on the association of the chemicals at stake. The major metabolic inhibitor observed was chlorfenvinphos. During co-incubation, it was able to decrease the intrinsic clearance of both linuron and ethion. These latter also showed a potential for metabolic inhibition mainly cytochrome P450-mediated in all cases. Here we demonstrated that human detoxification from a pesticide may be severely hampered in case of co-occurrence of other pesticides, as it is the case for drugs interactions, thus increasing the risk of adverse health effects. These results could contribute to improve the current challenging risk assessment of human and animal dietary to environmental chemical mixtures.

Comparable dynamics of linuron catabolic genes and IncP-1 plasmids in biopurification systems (BPSs) as a response to linuron spiking.

On-farm biopurification systems (BPSs) represent an efficient technology for treating pesticide-contaminated wastewater. Biodegradation by genetically adapted bacteria has been suggested to perform a major contribution to the removal of pesticides in BPSs. Recently, several studies pointed to the role of IncP-1 plasmids in the degradation of pesticides in BPSs but this was never linked with catabolic markers. Therefore, a microcosm experiment was conducted in order to examine whether changes in mobile genetic element (MGE) abundances in response to the application of phenylurea herbicide linuron are linked with changes in catabolic genes. Denaturing gradient gel electrophoresis (DGGE) fingerprints of 16S ribosomal RNA gene fragments amplified from total community (TC)-DNA suggested significant shifts in the bacterial community composition. PCR-Southern blot-based detection of genes involved in linuron hydrolysis (libA and hylA) or degradation of its metabolite 3,4-dichloroaniline (dcaQ I , dcaQ II , and ccdC) in TC-DNA showed that the abundance of the hylA gene was increased faster and stronger in response to linuron application than that of the libA gene, and that the dcaQ II gene was more abundant than the isofunctional gene dcaQ I 20 and 60 days after linuron addition. Furthermore, a significant increase in the relative abundance of the IncP-1-specific korB gene in response to linuron was recorded. Our data suggest that different bacterial populations bearing isofunctional genes coding for enzymes degrading linuron seemed to be enriched in BPSs in response to linuron and that IncP-1 plasmids might be involved in their dissemination.

Exploring the complex response to linuron of bacterial communities from biopurification systems by means of cultivation-independent methods.

On-farm biopurification systems (BPSs) treat pesticide-contaminated wastewater at farms through biodegradation and sorption processes. However, information on the microbiota involved in pesticide removal in BPSs is scarce. Here we report on the response of BPS bacterial communities to the herbicide linuron (BPS(+)) compared with the control (BPS(-)) in a microcosm experiment. Both denaturing gradient gel electrophoresis (DGGE) and pyrosequencing of 16S rRNA gene fragments amplified from community DNA indicated shifts in the bacterial community after linuron application. Responding populations belonged to taxa that were previously reported from linuron degrading consortia cultivated from soil (Hyphomicrobiaceae, Comamonadaceae, Micrococcaceae). In addition, numerous taxa with increased relative abundance were identified that were previously not associated with linuron degradation. The relative abundance of IncP-1 korB copies increased in response to linuron application. Amplicon pyrosequencing of IncP-1 trfA genes revealed a high IncP-1 plasmid diversity and suggested that populations carrying IncP-1ß plasmids increased in relative abundance. Transferable mercury resistance plasmids were exogenously captured from BPS(+)/BPS(-), and in three transconjugants from BPS(+) the gene hylA was detected. Our data suggest the existence of a multispecies linuron degrading bacterial food web and an involvement of IncP-1 plasmids in the adaptation of bacterial communities to pesticide pollution in BPSs.

Various effects of the photosystem II--inhibiting herbicides on 5-n-alkylresorcinol accumulation in rye seedlings.

The effect of three PSII-inhibiting herbicides, lenacil, linuron, and pyrazon, on the accumulation of 5-n-alkylresorcinols in rye seedlings (Secale cereale L.) grown under various light and thermal conditions was studied. All used chemicals increased resorcinolic lipid content in both green and etiolated plants grown at 29 °C. At 22 °C pyrazon and lenacil decreased the content of alkylresorcinols in plants kept in the darkness and increased their amount in the light-grown seedlings. In turn, level of resorcinolic lipids was decreased by linuron in both etiolated and green plants. At the lowest tested temperature lenacil enhanced production of alkylresorcinols only in etiolated rye seedlings, whereas the light-independent stimulatory action of pyrazon on alkylresorcinol accumulation in rye grown at 15 °C was observed. Additionally, only the latter did not exert a negative effect on rye seedling growth under any of tested conditions. Compared with respective controls, the herbicides used also markedly modified the qualitative pattern of resorcinolic homologs. Interestingly, the observed changes generally favored the enhanced antifungal activity of these compounds. Our study provides novel information on the influence of PSII inhibitors on alkylresorcinol metabolism in rye seedlings. The unquestionable achievement of this work is the observation that low dose of pyrazon mainly stimulated both growth and alkylresorcinol synthesis in rye seedlings, a non-target plant. Moreover, our experimental work showed unambiguously that the observed pyrazon-driven accumulation and homolog pattern modification of alkylresorcinols dramatically improved the resistance of winter rye to infections caused by Rhizoctonia cerealis.

Classifying chemical mode of action using gene networks and machine learning: a case study with the herbicide linuron.

The herbicide linuron (LIN) is an endocrine disruptor with an anti-androgenic mode of action. The objectives of this study were to (1) improve knowledge of androgen and anti-androgen signaling in the teleostean ovary and to (2) assess the ability of gene networks and machine learning to classify LIN as an anti-androgen using transcriptomic data. Ovarian explants from vitellogenic fathead minnows (FHMs) were exposed to three concentrations of either 5α-dihydrotestosterone (DHT), flutamide (FLUT), or LIN for 12h. Ovaries exposed to DHT showed a significant increase in 17ß-estradiol (E2) production while FLUT and LIN had no effect on E2. To improve understanding of androgen receptor signaling in the ovary, a reciprocal gene expression network was constructed for DHT and FLUT using pathway analysis and these data suggested that steroid metabolism, translation, and DNA replication are processes regulated through AR signaling in the ovary. Sub-network enrichment analysis revealed that FLUT and LIN shared more regulated gene networks in common compared to DHT. Using transcriptomic datasets from different fish species, machine learning algorithms classified LIN successfully with other anti-androgens. This study advances knowledge regarding molecular signaling cascades in the ovary that are responsive to androgens and anti-androgens and provides proof of concept that gene network analysis and machine learning can classify priority chemicals using experimental transcriptomic data collected from different fish species.

Using additive modelling to quantify the effect of chemicals on phytoplankton diversity and biomass.

Environmental authorities require the protection of biodiversity and other ecosystem properties such as biomass production. However, the endpoints listed in available ecotoxicological datasets generally do not contain these two ecosystem descriptors. Inferring the effects of chemicals on such descriptors from micro- or mesocosm experiments is often hampered by inherent differences in the initial biodiversity levels between experimental units or by delayed community responses. Here we introduce additive modelling to establish the effects of a chronic application of the herbicide linuron on 10 biodiversity indices and phytoplankton biomass in microcosms. We found that communities with a low (high) initial biodiversity subsequently became more (less) diverse, indicating an equilibrium biodiversity status in the communities considered here. Linuron adversely affected richness and evenness while dominance increased but no biodiversity indices were different from the control treatment at linuron concentrations below 2.4 µg/L. Richness-related indices changed at lower linuron concentrations (effects noticeable from 2.4 µg/L) than other biodiversity indices (effects noticeable from 14.4 µg/L) and, in contrast to the other indices, showed no signs of recovery following chronic exposure. Phytoplankton biomass was unaffected by linuron due to functional redundancy within the phytoplankton community. Comparing thresholds for biodiversity with conventional toxicity test results showed that standard ecological risk assessments also protect biodiversity in the case of linuron.

The effects of the urea-based herbicide linuron on reproductive endpoints in the fathead minnow (Pimephales promelas).

Linuron is a widely used urea-based herbicide that has anti-androgenic activity in both fish and rodents. To further elucidate the potential mode of action (MOA) of linuron on the vertebrate endocrine system, adult male and female fathead minnows were exposed for 21 days to dechlorinated water, a solvent control, 17ß-estradiol (E2; 0.1 µg/L), dihydrotestosterone (DHT; 100 µg/L), linuron (1, 10, 100 µg/L) and one co-treatment of DHT (100 µg/L) and linuron (100 µg/L). There were no effects of linuron on egg hatching, 7 day egg survival, nuptial tubercle formation or gonadal histopathology. Administration of DHT and 1 and 100 µg/L linuron reduced plasma vitellogenin in females, while male plasma vitellogenin were induced after E2 exposure and co-exposure of DHT and linuron. Ovarian mRNA levels were examined for several genes involved in steroidogenesis (e.g. p450scc, cyp19a, star, tspo, hsd17b and hsd11b) and estrogen-mediated responses (esr1, esr2b, esr2a). Only p450scc mRNA was significantly decreased with DHT+linuron co-treatment. Clustering of steroidogenic mRNA transcript expression patterns revealed that patterns for linuron were more similar to E2 compared to DHT. Collectively, this study supports the hypothesis that linuron may not be a pure anti-androgen and may have multiple MOAs that affect vertebrate reproduction.

[Development of corrosion microbial association of soil in the presence of linuron and its derivatives].

Qualitative stability of corrosion-dangerous microbial association, extracted from ferrosphere of corroding underground pipelines in different soils is shown. Prevailing of sulphate-reducing and iron-reducing bacteria is peculiar to the structure of formed corrosion-aggressive microbial cenosis. Development of association of iron-reducing bacteria in the presence of acting matter of pesticide Linuron and its derivatives is depressed. The association of sulphate-reducing bacteria is sensitive only to the derivate that contains triazolazepine cycle and tolyl.

Estradiol and resveratrol stimulating effect on osteocalcin, but not osteonectin and collagen-1alpha gene expression in primary culture of rat calvarial osteoblast-like cells.

Evidence is available that some endocrine disruptors, acting as selective estrogen receptor modulators (SERMs), interfere with osteoblast differentiation and function. Therefore, we investigated whether 17beta-estradiol, bisphenol-A (BSP), silymarin, genistein, resveratrol, procymidone, linurone and benzophenone-3 (BP3) modulate differentiation of rat calvarial osteoblast-like (ROB) cells in primary in vitro culture. Disruptors were added at day 18 of culture and cells were harvested 48 h later. Real time-PCR revealed that estradiol and resveratrol enhanced osteocalcin mRNA expression in ROB cells, while other disruptors were ineffective. The expression of osteonectin and collagen-1alpha was not affected by any disruptor. Estradiol, resveratrol, genistein and BSP stimulated the proliferative activity of ROB cells. In contrast, procymidone and linurone inhibited the proliferative activity, and silymarin and BP3 were ineffective. The conclusion is drawn that i) only resveratrol is able, like estradiol, to stimulate the specialized functions of ROB cells, and ii) the proliferative activity of ROB cells is more sensitive to endocrine disruptors, some of which could probably act via a mechanism independent of their SERM activity.

Effect of pre-emergence herbicides on growth parameters of green pea.

Green pea (Pisum sativum L.), is one of the important vegetable crop in Hungary. Chemical weed control has an important role in pea growing. Pre-emergence herbicides are used most frequently in a green pea culture because they eliminate competition between crop plant and weeds even at the critical early growth stage. Preemergence treatment combined with mechanical methods makes unnecessary the post-emergence protection. Herbicides with different mode of action can influence the growth of pea and cause phytotoxic symptoms. Sensitivity of plant varieties against herbicides is different in the first place due to the thickness ofleave's wax layer. The aim of our experience was to study the effect of five pre-emergence herbicides with different mode of action on the fresh and dry matter production and growing of a green pea variety (Pisum sativum cv. Petit Provencal). The examined herbicides were Pivot (imazethapyr), Proponit 840 EC (propisochlor), Sencor 70 WG (metribuzin), Stomp 330 (pendimethalin), and Afalon Dispersion (linuron). Pot experiments were carried out under greenhouse conditions in four replications. Herbicides were applied in the suggested and double doses. Four weeks after sowing the length, fresh- and dry weight of the shoots and the roots of pea were determined. It was an interesting observation that Pivot raised fresh weight significantly. Sencor 70 WG caused the most dramatic fresh mass reduction. We have got similar tendencies in dry matter production of green pea samples. Double rate of Stomp 330 and Sencor 70 WG decreased length of shoots and roots significantly. We established that examined pre-emergence herbicides could influence growth parameters to a different extent. Sencor 70 WG strongly inhibited growth of pea and caused severe phytotoxic symptoms. Double dose of Proponit 840 EC and Stomp 330 also damaged the pea but to a lesser extent. Afalon Dispersion and Pivot proved to be the most suitable herbicides; pea wasn't sensitive to these chemicals. Due to joining to EU permission of Pivot was cancelled in Hungary.

Effects of bisphenol-A (BPA), dibutylphtalate (DBP), benzophenone-2 (BP2), procymidone (Proc), and linurone (Lin) on fat tissue, a variety of hormones and metabolic parameters: a 3 months comparison with effects of estradiol (E2) in ovariectomized (ovx) rats.

The endocrine active substances BPA, DBP and BP2 have estrogenic effects in the uterus. Proc and Lin were shown to be antiandrogenic. Whether other estrogen-regulated parameters like lipids, fat metabolism and hormones are also affected by these substances is unknown. We compared the effects of a 3 months lasting administration of these substances with those of E2 on an estrogen-regulated fat depot and on serum TSH, T3, T4, LH, and lipid concentrations. BP2 shared many of the effects of E2 by suppressing LH, cholesterol, LDL, HDL and the size of a small estrogen-regulated fat depot in the lower hindleg. BP2 suppressed T4 but not T3 and TSH. The effects of DBP and BPA were mostly negligible whereas Lin and Proc also reduced the fat depot and serum leptin but increased triglycerides and serum lipids. Serum T3 and T4 concentrations were reduced by Lin and Proc. These data indicate that BP2 has similar, though slightly weaker effects as E2 whereas the effects of Proc and Lin differ from those of E2 on serum lipids and hormones. BP2 can be regarded as an estrogenic endocrine active substance whereas the effects of Lin and Proc appear not to involve estrogen receptors.

A mixture of the "antiandrogens" linuron and butyl benzyl phthalate alters sexual differentiation of the male rat in a cumulative fashion.

Prenatal exposure to environmental chemicals that interfere with the androgen signaling pathway can cause permanent adverse effects on reproductive development in male rats. The objectives of this study were to 1) determine whether a documented antiandrogen butyl benzyl phthalate (BBP) and/or linuron (an androgen receptor antagonist) would decrease fetal testosterone (T) production, 2) describe reproductive developmental effects of linuron and BBP in the male, 3) examine the potential cumulative effects of linuron and BBP, and 4) investigate whether treatment-induced changes to neonatal anogenital distance (AGD) and juvenile areola number were predictive of adult reproductive alterations. Pregnant rats were treated with either corn oil, 75 mg/kg/day of linuron, 500 mg/kg/day of BBP, or a combination of 75 mg/kg/day linuron and 500 mg/kg/day BBP from gestational Day 14 to 18. A cohort of fetuses was removed to assess male testicular T and progesterone production, testicular T concentrations, and whole-body T concentrations. Male offspring from the remaining litters were assessed for AGD and number of areolae and then examined for alterations as young adults. Prenatal exposure to either linuron or BBP or BBP + linuron decreased T production and caused alterations to androgen-organized tissues in a dose-additive manner. Furthermore, treatment-related changes to neonatal AGD and infant areolae significantly correlated with adult AGD, nipple retention, reproductive malformations, and reproductive organ and tissue weights. In general, consideration of the dose-response curves for the antiandrogenic effects suggests that these responses were dose additive rather than synergistic responses. Taken together, these data provide additional evidence of cumulative effects of antiandrogen mixtures on male reproductive development.

Oxidative stress and bioindicators of reproductive function in pulp and paper mill effluent exposed white sucker.

This study investigates oxidative stress and bioindicators of reproductive function in wild white sucker (Catostomus commersoni) collected from environments receiving pulp and paper mill effluent discharges in northern Ontario. Samples were collected over an eight-year period adjacent to three pulp and paper mills using a variety of processing and bleaching techniques. Fish collected downstream of pulp and paper mills within the Moose River basin exhibited elevated hepatic and gonadal 2-thiobarbituric acid reactive substances (TBARS), the presence of which is indicative of oxidative stress in these tissues. Within the Jackfish Bay system, exposure to pulp and paper mill effluent did not elevate hepatic or gonadal TBARS. Hepatic cytochrome P4501A activity (CYP1A) and fatty acyl-CoA oxidase (FAO) activities were frequently increased in livers of Moose River basin fish exposed to pulp and paper mill effluent, while lower activities of both enzymes were found within fish from the Jackfish Bay system. This suggests that oxidative stress may be related to CYP1A and FAO activities. Within the Moose River system, increases in measures of oxidative stress (TBARS, FAO) were generally coincident with decreased levels of 17 beta-estradiol; however, testosterone was often lower in Jackfish Bay system fish without any commensurate changes in oxidative stress. The suite of reproductive and oxidative stress parameters measured in this study varied between seasons and mills suggesting responses to effluent are dynamic and effects are complicated by different receiving environments. The relationship between gonad size, gonadal oxidative stress, and circulating plasma steroids remains unclear.